From owner-proteins@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!agate!newsfeed.kornet.nm.kr!howland.erols.net!newsfeed.nacamar.de!univ-lyon1.fr!citi2.fr!federici.cochin.inserm.fr!user From: federici@icgm.cochin.inserm.fr (Federici) Newsgroups: bionet.molbio.proteins Subject: Re: preventing protein degradation Date: 2 Oct 1997 12:10:09 GMT Organization: ICGM CNRS UPR415 Lines: 27 Message-ID: References: <19970925020801.WAA16004@ladder02.news.aol.com> NNTP-Posting-Host: federici.cochin.inserm.fr X-Newsreader: Yet Another NewsWatcher F2.0 In article (Dans l'article) <19970925020801.WAA16004@ladder02.news.aol.com>, yankiwski@aol.com (Yankiwski) wrote (écrivait) : > Hi, > > I'm expressing a protein in Sf9 insect cells using a baculovirus vector. > Upon purification of the 6x his-tagged protein on a nickel affinity column > and after Western analysis, I get multiple bands, with the majority being > of smaller size and in greater quantity than the full-length protein. This > is the case in both denaturing and nondenaturing purification conditions, > and is not ameliorated by the use of "protease inhibitor cocktail" > tablets, PMSF, and beta-mercaptoethanol in the lysis buffer and in > subsequent wash and elution steps. Assuming specificity of the Ab, and > that the multiple bands are in fact due to degradation, what can be done to > remedy the problem? > > Thanks for any suggestions. > Victor To assume specificity of antibody and be sure the bands are due to degradation you must recuperate one of these bands and sequence it. You must have in mind that the bands are due to non specific reactions; Instead of using in the first step the nickel affinity column, I reckon another purification step, in denaturing conditions, must be performed before Ni column (gel filtration, menbrane filtration,or hydroxyapatite chromato.) From owner-proteins@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!agate!hammer.uoregon.edu!csulb.edu!logbridge.uoregon.edu!news-peer.gsl.net!news.gsl.net!gip.net!howland.erols.net!recycled.news.erols.com!news.uni-c.dk!balder.adm.ku.dk!arnold!tlk From: Toke Lindegaard Knudsen Newsgroups: bionet.molbio.proteins Subject: Breaking the barriers between biology, chemistry and physics. Date: Thu, 2 Oct 1997 15:29:21 +0200 Organization: University of Copenhagen Lines: 356 Message-ID: NNTP-Posting-Host: arnold.math.ku.dk Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII X-Sender: tlk@arnold I am submitting this very interesting article on behalf of Madhavendra Puri of the Bhaktivedanta Institute. Could anyone replying to this posting on the newsgroup please CC a copy to Madhavendra Puri at . That would be most helpful for me. Thank you! -Toke Lindegaard Knudsen. EVIDENCE THAT ATOMS BEHAVE DIFFERENTLY IN BIOLOGICAL SYSTEMS THAN OUTSIDE OF THEM Madhavendra Puri The Bhaktivedanta Institute E-mail: tumle@diku.dk A number of chemists report that plants, animals and human beings ROUTINELY TRANSMUTE MID-RANGE ELEMENTS (for example, potassium into calcium or magnesium into calcium) AS PART OF THEIR ORDINARY DAILY METABOLISM. These transmutations obey rules such as: Mg + O => Ca; K + H => Ca. This is revolutionary since, according to current physical theory, the energy levels required for such transmutations are billions of times higher than what is available in biological systems. Equally inexplicable fission reactions such as Ca => Mg + O; Ca => K + H are also reported. But revolutions in physics have repeatedly occurred, such as the quantum revolution in which the radical property of non-locality, previously considered impossible, is now accepted by physicists (see Aspect and Grangier 1986, Bransden and Joachain 1989, p.671-681, Chiao et al 1993, Squires 1990, p.173, Rae 1986, p.25-44, and Penrose 1990, p.369). What I am presenting here is not the "cold fusion" of Fleischmann and Pons which, as far as I know, lacks clear evidence of actual fusion. Even if the Fleischmann and Pons effect turns out to be actual fusion, it is only the fusion of isotopes of the lightest element hydrogen under special laboratory conditions which is quite different from the UNEQUIVOCAL FUSION AND FISSION OF MID-RANGE elements found in biological transmutation reports. Now let us examine the evidence for biological transmutation. Crabs, shellfish and crayfish have shells made largely of calcium. A crab 17 cm by 10 cm has a shell weighing around 350 grams. Periodically these animals shed their shell and create a new one. This is called molting. When molting, a crab is very vulnerable and hides away from all other creatures so it can not get calcium by preying on other creatures. According to French chemist C. Louis Kervran of the Conseil d'Hygiene in Paris, sea water contains far too little calcium to account for the rapid production of a shell (the calcium content of sea water is about 0.042% and a crab can form a new shell in little more than one day). If the entire body of a crab is analyzed for calcium, it is found to contain only enough calcium to produce 3% of the shell (even taking into account the calcium carbonate stored in the hepato-pancreas just before molting). Even in water completely devoid of calcium, shellfish can still create their calcium-bearing shells as shown by an experiment performed at the Maritime Laboratory of Roscoff: "A crayfish was put in a sea water basin from which calcium carbonate had been removed by precipitation; the animal made its shell anyway." (Kervran 1972, p.58) "Chemical analysis made on animals secreting their shells has revealed that calcium carbonate is formed on the outer side of a membrane although on the opposite side of the membrane, where matter enters, there is no calcium. This fact has left specialists perplexed." (Kervran 1972, p.58) Sea water contains a sufficient amount of magnesium to form a shell if we accept Kervran's proposition that crabs routinely transmute magnesium into calcium; Mg + O => Ca. It would be interesting to put a crayfish in water devoid of both calcium and magnesium and see if it can still create its shell. Normal egg shells produced by hens contain calcium. Kervran (1972, p.41) reported an experiment in which hens were confined in an area in which there was no source of calcium and no calcium was present in their diet. The calcium deficiency became clearly manifested after a few days when the hens began to lay eggs with soft shells. Then purified mica (which contains potassium) was given to the hens. Kervran (1972, p.41) described what then transpired: "The hens jumped on the mica and began scratching around it very rapidly, panting over it; then they rested, rolling their heads on it, threw it into the air, and began scratching it again. The next day eggs with normal shells (weight 7 grams) were laid. Thus, in the 20 hours that intervened, the hens transformed a supply of potassium into calcium. ... An experiment of this kind, using the same mica, was undertaken with guinea-fowls over a period of forty days. The administering of the mica was suspended three times and each time a soft-shelled egg was laid ... ." One might suggest that the calcium in the egg shells was borrowed from the bones of the hens. But if this is true, why were soft eggs laid when the mica was withheld and normal eggs laid when mica was given to the hens? In order to avoid the conclusion that the hens transmuted potassium into calcium, one would have to show that mica somehow stimulates a metabolic pathway in which calcium is removed from the hen's bones and used in the production of the egg shells. This could be completely refuted by feeding the hens mica (and of course absolutely no calcium) for such a long period of time that all the calcium in their bones would have been completely exhausted. If after that time the hens still produce calcium-bearing egg shells, we must conclude that the calcium in the egg shells is not being taken from the bones. At that point, we seem to have no choice but to acknowledge the transmutation of potassium into calcium within the hens. Kervran (1972, p.52) described experiments performed in 1959 by the French government in the Sahara desert. The government was interested in determining the nutritional requirements of petroleum workers in the extreme heat prevalent in the desert. In the first experiment, conducted near a place called Ouargla, the total amount of magnesium ingested per day per man was measured and compared with the amount excreted. It was found that, on the average, each man daily excreted 117.2 milligrams of magnesium more than he ingested. Thus, each day, each man lost on the average 117.2 milligrams of magnesium. Now we must consider how much magnesium is on reserve in the human body: it turns out that the body is not able to mobilize more than 5000 milligrams of magnesium. Thus, at a daily loss of 117.2 milligrams, it is clear that after 50 days the bodies of the petroleum workers should have been completely depleted of magnesium. But the experiment was conducted for 180 days and each day each man excreted on the a verage 117.2 milligrams more than he ingested. The second experiment lasted for 240 days and was conducted near Tindouf which has a drier climate. This time each man excreted each day an average of 256 milligrams of magnesium more than he ingested. Under these conditions, after 20 days, each man should have been completely depleted of magnesium; but somehow they survived for 220 days thereafter. It seems difficult to avoid the conclusion that the human body is able to create magnesium. Biochemist H. Komaki of the University of Mukogawa in Japan reported that a number of different families of microorganisms such as Aspergillus niger and Saccharomyces cerevisiae create potassium during growth. (Komaki 1965, 1967) Kervran described a germination experiment using ryegrass seeds (type Rina) performed in 1971 by the Laboratory of the Societe des Agriculteurs de France (Kervran 1972, p.107). Out of an initial group of 2000 seeds, 1000 were set aside as a control batch and the other 1000 were germinated. The control batch weighed 2.307 grams before drying and 2.035 grams after drying. These 2.035 grams were analyzed and found to contain 3.02 milligrams of magnesium, 6.97 milligrams of potassium, 6.00 milligrams of calcium and 0.021 milligrams of copper. The magnesium, calcium and copper contents were determined by atomic absorption spectroscopy and the potassium content was determined by flame emission. The 1000 seeds to be germinated were germinated for 29 days in Petri dishes under a plastic sheet to insure that no dust could get in. Aside from 430 milliliters of Evian water, absolutely nothing else was supplied to the seeds during germination. 430 milliliters of Evian water was found to contain 10.32 milligrams of magnesium, 0.39 milligrams of potassium, 33.11 milligrams of calcium and 0.00 milligrams of copper. After the 29 day germination period, the plants were converted to ashes under high temperature and the ashes and residual Evian water in the Petri dishes were found to contain 3.20 milligrams of magnesium, 16.67 milligrams of potassium, 36.50 milligrams of calcium and 0.10 milligrams of copper. Before germination there were 6.97 milligrams of potassium in the seeds. During germination 0.39 milligrams of potassium were added to the growing plants (this came from the Evian water). If atomic nuclei can not be altered in biological systems, we expect that after germination there should be 6.97 + 0.39 = 7.36 milligrams of potassium in the plants and residual Evian water. But this was not the case. After germination the plants and residual Evian water were found to contain 16.67 milligrams of potassium. Thus 9.31 milligrams of potassium were apparently created during germination. Before germination there were 3.02 milligrams of magnesium in the seeds. During germination 10.32 milligrams of magnesium were added to the growing plants (this came from the Evian water). If atomic nuclei can not be altered in biological systems, we expect that after germination there should be 10.32 + 3.02 = 13.34 milligrams of magnesium in the plants and residual Evian water. But after germination the plants and residual Evian water were found to contain only 3.20 milligrams of magnesium. Thus 10.14 milligrams of magnesium were apparently destroyed during germination. Before germination there were 0.021 milligrams of copper in the seeds. During germination 0.00 milligrams of copper were added to the growing plants. Assuming that atomic nuclei can not be altered, we expect that after germination there should still be 0.021 milligrams of copper in the plants and residual Evian water. But it turned out that after germination the plants and residual Evian water were found to contain 0.10 milligrams of copper. Thus 0.079 milligrams of copper were apparently created during germination. Before germination there were 6.00 milligrams of calcium in the seeds. During germination 33.11 milligrams of calcium were added to the growing plants (from the Evian water). Assuming that nuclei can not be altered, we expect that after germination there should be 39.11 milligrams of calcium in the plants and residual Evian water. However, after germination the plants and residual Evian water were found to contain 36.50 milligrams of calcium. Thus 2.61 milligrams of calcium were apparently destroyed during germination. The following challenge can be made: no one knows how much potassium, calcium, magnesium and copper was in the seeds before they were germinated. It was assumed that the amounts of these elements was not significantly different from the amounts of these elements in the control batch. How do we know this is true? What should have been done is to start with a 100 grams of seeds, mix them around thoroughly, weigh out 50 batches of 2.000 grams each, randomly select 25 of these as control batches, determine the amounts of potassium, calcium, magnesium and copper in these batches and note the maximum variation in these elements among these batches. The remaining 25 batches can then be germinated and the plants analyzed for element content. In this way we would have some measure of the variation among different batches (both germinated and control). On the positive side, it can be argued that since the seeds of the control and germinated batches were of the same type, the variation in element content between these two batches was not significant. Some support for this idea can be found in the data provided by chemist D. Long of the Michaelis Nutritional Research Laboratory in Harpenden, England. Long analyzed (using atomic spectroscopy) six batches of ryegrass seeds (each of which weighed 5.4 grams before drying) and discovered that the difference in potassium content between the batch containing the greatest amount of potassium and the batch containing the least amount of potassium was 0.054 milligrams of potassium per gram of dry seed weight. Similarly, the maximum difference in magnesium content was 0.033 milligrams per gram of dry seed weight, that of calcium was 0.091 milligrams per gram of dry seed weight, and that of copper was 1.19 micrograms per gram of dry seed weight. (Long 1971, p.7) Kervran proposed that the plants performed the following nuclear reactions: Mg + O => Ca; Ca => K + H. Kervran did not discuss the reaction involving copper. Based on experience derived from similar experiments, Kervran said that if the seeds are germinated in doubly-distilled water, the amount of transmuted material is much smaller and may fall within the range of experimental error and therefore not be significant. The reason for this is that each kind of plant is only able to transmute certain elements into certain other elements. Thus the experimenter must provide the plant with a certain amount of certain elements if he wants to observe a large amount of transmuted material. For germinating ryegrass seeds, Evian water is the perfect growth medium because it provides this particular kind of plant with the elements it needs. Kervran (1972, p.132) also described a series of experiments in which wheat and oat seeds were germinated "on porous ashless paper saturated with a fertilizing solution of salts dissolved in water. The solution was free of calcium." In the case of wheat (Roux Clair) there was 3.34 times more calcium in the plants than in the seeds; in the case of one kind of oats (Noire du Prieure) there was 4.16 times more calcium in the plants than in the seeds; in the case of another kind of oats (Panache de Roye) there was 4.51 times more calcium in the plants than in the seeds. The calcium content was determined by two independent methods (conventional chemical analysis and atomic absorption spectroscopy); both methods agreed closely. Kervran performed more than 20 such experiments, mostly on oat seeds. Kervran (1972, p.133) mentioned that the moon plays an important role in the production of calcium. The above huge increases in calcium were obtained in experiments in which the germination started at the new moon and stopped on the second full moon (after 6 weeks). This is an important consideration for those who attempt to duplicate these results. A lunar influence on the metabolic activity of various plants and animals was also reported by biologist Frank A. Brown. (Gauquelin 1969, p.131-133) D. Long questioned Kervran's methods of analysis. Long (1971, p.9) said that Kervran had made (in some of his earlier experiments) the mistake of comparing the ash weight of the control batch with the ash weight of the plants after germination. Kervran may have made this mistake in some of his earlier experiments but he did not do so in the ryegrass, wheat and oat germination experiments described above. In these experiments, he rightly compared the weight of the control batch with the weight of the seeds to be germinated. In other words, the weight comparison was done on the two batches of seeds before one batch was germinated. This is the correct procedure as acknowledged by Long himself. Long germinated ryegrass seeds in deionized water and reported that he was unable to observe a transmutation of elements. As discussed above, this is to be expected since without a sufficient input of certain elements, there is insufficient material to be transmuted. A more serious criticism is Long's claim that he corresponded with Kervran who advised him to germinate green lentil seeds (Leguminacae) in water containing certain minerals. Long reported that although he did this he was still unable to observe a significant transmutation of elements. But Long did not attempt to duplicate the best of Kervran's germination experiments, namely the ryegrass, wheat and oat experiments described above. I hope that many scientists will do these experiments and report the results to the scientific community. In the 1950s Pierre Baranger, a professor and the director of the Laboratory of Organic Chemistry at the Ecole Polytechnique in Paris, performed a large number of germination experiments and concluded that plants routinely transmute elements. Baranger did his experiments independently of Kervran. Baranger said: "My results seem impossible, but here they are. I took every precaution. I repeated the experiments many times. I made thousands of analyses for years. I had the results verified by third parties who did not know what I was investigating. I used several methods. I changed my experimenters. But there is no escape. We must submit to the evidence: plants transmute elements." (Michel 1959, p.82) I tried to get more information by writing letters to the Ecole Polytechnique, the Societe des Agriculteurs de France and the Agronomie Research National Institute, but I received no reply. In 1975 chemists O. Heroux and D. Peter of the Division of Biological Sciences of the National Research Council of Canada conducted a meticulous experiment with rats (Heroux and Peter 1975). They measured the amount of magnesium ingested through food, water (and even air) as well as the amount of magnesium excreted in the form of urine and feces over three periods of time: 69 days, 240 days and 517 days. In the case in which the rats were fed a diet in which the amount of magnesium ingested was less than the amount of magnesium excreted, it was expected that the total amount of magnesium in the body would decrease. In fact, long before the 517th day of the experiment it was expected that there would be zero magnesium in the body. However, when the rats were analyzed for total magnesium on the 517th day, each rat contained, on the average, 82 milligrams of magnesium. The method used to determine the amount of magnesium in the body, food, water, air, feces and urine was atomic absorption spectroscopy. Heroux and Peter verified the accuracy of their determinations by giving samples to two other laboratories (the Division of Chemistry at the National Research Council and the Department of Chemistry at McMaster University); both of these laboratories obtained essentially the same results as Heroux and Peter at the Division of Biology at the National Research Council. Finally, other methods were used (such as destructive neutron activation and spectrographic emission) and these methods yielded results very similar to those obtained using atomic absorption spectroscopy. I do not advise the replication of this experiment since it involved killing the rats in order to analyze their bodies for magnesium. Experiments involving animal killing are not required since there are many ways (as described above) to verify biological transmutation without such killing. Bibliography Albert, D. "Bohm's Alternative to Quantum Mechanics." Scientific American, May 1994, pages 32-39 Aspect, A. and Grangier, P. "Experiments on Einstein- Podolsky-Rosen-type Correlations with Pairs of Visible Photons." In Quantum Concepts in Space and Time (edited by R. Penrose and C. J. Isham). Oxford: Oxford University Press, 1986 Bohm, D. and Peat, F. Science, Order and Creativity. New York: Bantam Books, 1987 Bransden, B. and Joachain, C. Introduction to Quantum Mechanics. Essex: Longman Group U.K. Limited, 1989 Chiao, R., Kwait, P. and Steinberg, A. "Faster than light?" Scientific American, August 1993, pages 38-46 Darnell, J., Lodish, H. and Baltimore, D. Molecular Cell Biology. New York: W. H. Freeman and Co., 1990 Gauquelin, M. The Cosmic Clocks. London: Peter Owen, 1969 Heroux, O. and Peter, D. "Failure of balance measurements to predict actual retention of magnesium and calcium by rats as determined by direct carcass analysis." Journal of Nutrition, 1975, volume 105, pages 1157-1167 Kervran, C. Louis. Biological Transmutation. New York: Swan House Publishing Company, 1972 Komaki, H. "Sur la formation de sels de potassium par differentes familles de microorganismes dans un milieu sans potassium." Revue de Pathologie Comparee, Paris, September 1965 Komaki, H. "Production de proteines par 29 souches de microorganismes et augmentation du potassium en milieu de culture sodique, sans potassium." Revue de Pathologie Comparee, Paris, April 1967 Long, D. B. "Laboratory Report on Biological Transmutation." Monograph of the Henry Doubleday Research Society. Braintree, Essex, England, September 1971 Michel, A. "Un savant francais bouleverse la science atomique." Science et Vie, Paris, 1959, pages 81-87 Penrose, R. The Emperor's New Mind. New York: Vintage Press, 1990 Rae, A. Quantum Physics: Illusion or Reality? Cambridge: Cambridge University Press, 1986 Squires, E. Conscious Mind in the Physical World. Bristol: Adam Hilger, 1990 From owner-proteins@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!agate!howland.erols.net!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.internetmci.com!152.163.199.19!portc03.blue.aol.com!audrey02.news.aol.com!not-for-mail From: huntpharm@aol.com (Huntpharm) Newsgroups: bionet.molbio.proteins Subject: US-NY-Ph.D. JOB OPPORTUNITY IN PROTEIN PURIFICATION Date: 2 Oct 1997 11:40:53 GMT Lines: 17 Message-ID: <19971002114000.HAA29457@ladder02.news.aol.com> NNTP-Posting-Host: ladder02.news.aol.com X-Admin: news@aol.com Organization: AOL http://www.aol.com I am looking for a Scientist to perform analytical characterization of proteins including N-Terminal sequencing, disulfide mapping of proteins, and mass spectometry. You will be responsible for all aspects of small scale bio-analytical characterization of proteins from recombinant sources. You will also be responsible for technologies including N-Terminal sequencing, amino acid analysis, glycosylation analysis, disulfide mapping of proteins, as well as epitope mapping of proteins. This person should have experience in protein purification and characterization. The candidate will have 2+ years post-doctoral experience and possess a Ph.D. in Biochemistry, Biology, Biophysics, Organic Chemistry or related field. We are a leading biotech firm with research facilities in Tarrytown, New York and can provide excellent benefits (health insurance, dental, and vision plan, paid vacation and more). A high impact, high profile position with excellent opportunity for advancement. Please contact Scott Shanes by phone at 609-584-8733 Ext. 218, fax CV and cover letter to 609-584-9575 or E-Mail to sis@dmc10.com or sis@diedremoire.com From owner-proteins@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!SIRIUS.COM!lancelot From: lancelot@SIRIUS.COM (Lance Wong) Newsgroups: bionet.molbio.proteins Subject: Deglycosylation Date: 2 Oct 1997 12:34:48 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 3 Sender: daemon@net.bio.net Distribution: world Message-ID: <343307F8.686E@sirius.com> Reply-To: lancelot@sirius.com NNTP-Posting-Host: net.bio.net Thank you for pointing me to this site. I found the booklet very informative, especially about removing O-linked sugars. The references were also useful. From owner-proteins@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!agate!hammer.uoregon.edu!csulb.edu!logbridge.uoregon.edu!newsfeed.internetmci.com!194.162.162.196!newsfeed.nacamar.de!univ-lyon1.fr!cnusc.fr!ciril.fr!univ-angers.fr!univ-rennes1.fr!not-for-mail From: Myriam.Onno@univ-rennes1.fr (Myriam Onno) Newsgroups: bionet.molbio.proteins Subject: mAbs against golgi and ER Date: 2 Oct 1997 16:44:36 GMT Organization: Universite de Rennes1 Lines: 9 Message-ID: <610j1k$684$2@news.univ-rennes1.fr> NNTP-Posting-Host: pc-hemato.univ-rennes1.fr Mime-Version: 1.0 Content-Type: Text/Plain; charset=ISO-8859-1 X-Newsreader: WinVN 0.99.5 I am going to precise the localization of a protein in the cell Does it exist monoclonal antibodies against the golgi apparatus and the endoplasmic reticulum ? Thanks From owner-proteins@net.bio.net Wed Oct 01 23:00:00 1997 Path: biosci!agate!newsfeed.kornet.nm.kr!news.maxwell.syr.edu!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!gatech!news-feed-1.peachnet.edu!cronkite.cc.uga.edu!cronkite.cc.uga.edu From: Margie Lee Newsgroups: bionet.molbio.proteins Subject: Qiagen anti-His tag antibody Date: Thu, 02 Oct 1997 18:47:23 -0400 Organization: uga,cvm Lines: 6 Message-ID: <3434247B.B51@calc.vet.uga.edu> NNTP-Posting-Host: s28-pm03.athens.campus.mci.net Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01Gold (Win95; I) We've also had problems with that antibody. One of the problems is the antibody is a strange variant of the isotype stated in the package insert. We tried 4 secondary antibodies (antiIgG1) to find one that would detect the Qiagen product. But the antibody does not detect our His-tagged protein (N-term 6 histidines) which purifies easily on the column. From owner-proteins@net.bio.net Thu Oct 02 23:00:00 1997 Path: biosci!rutgers!nntp.upenn.edu!dsinc!spool.mu.edu!uwm.edu!vixen.cso.uiuc.edu!howland.erols.net!newsfeed.internetmci.com!204.238.120.130!jump.net!grunt.dejanews.com!not-for-mail Date: Fri, 03 Oct 1997 23:01:20 -0600 From: jorges@bcm.tmc.edu Subject: Re: Anti-His tag antibodies Newsgroups: bionet.molbio.proteins Message-ID: <875764386.1149@dejanews.com> Organization: Deja News Posting Service References: <199709252033.PAA25676@mis.finchcms.edu> X-Article-Creation-Date: Thu Oct 02 03:53:07 1997 GMT X-Originating-IP-Addr: 128.249.96.100 () X-Http-User-Agent: Mozilla/3.0Gold (WinNT; I) X-Authenticated-Sender: jorges@bcm.tmc.edu Lines: 17 I have not yet tried any of the His-antibodies but there is an antibody from Invitrogen specifically against C-terminal His. From the online catalogue "The Anti-His(C-term) Antibody is a purified monoclonal antibody that detects the following as an epitope: -(oligohistidine)-His-COOH (minimal number of histidines and additional amino acids required is not precisely defined) This antibody can be used to detect fusion proteins expressed from vectors such as pSecTag, which incorporates a C-terminal polyhistidine tag as a fusion with the expressed recombinant protein. " No connection with Invitrogen, etc. -------------------==== Posted via Deja News ====----------------------- http://www.dejanews.com/ Search, Read, Post to Usenet From owner-proteins@net.bio.net Sat Oct 04 23:00:00 1997 Path: biosci!agate!spool.mu.edu!uwm.edu!vixen.cso.uiuc.edu!news-peer.sprintlink.net!news-pull.sprintlink.net!news-in-east.sprintlink.net!news.sprintlink.net!Sprint!193.10.88.100!news00.sunet.se!sunic!mn6.swip.net!mn5.swip.net!news From: Stephan Schoen Newsgroups: bionet.molbio.proteins Subject: Ni-NTA HisSorb Strips Date: Sun, 05 Oct 1997 16:23:40 +0200 Organization: A customer of Tele2 Lines: 31 Message-ID: <3437A2EC.418D23E9@genetics.su.se> NNTP-Posting-Host: mn8.swip.net Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Mailer: Mozilla 4.02 [en] (Win95; I) Cache-Post-Path: mn8!s-246439@dialup161-2-54.swipnet.se hi, we are planning to use Qiagen Ni-NTA HisSorb Strips to look for protein-protein interaction. Unfortunately, there are hardly any protocols provided with the strips, none of them for protein-protein interaction. Contacting Qiagen didn´t help either, although they mention protein-protein interaction in their catalog, it was "an observation in the lab" (quote) rather than a planned feature. Therefore, they don´t have any helpful information or protocols for us. The initial idea was to use the strips to bind our his-tagged protein to the wells, add the second interacting protein, wash, add antibody, wash, add secondary antibody, wash, detect (kind of like Elisa). I was wondering if anyone out there has ever used these plates, has done something similar or knows any reference to this problem, using HisSorb Strips or Elisa plates to look for protein-protein interaction. any comments or ideas are welcome, thanx, stephan --- stephan@genetics.su.se From owner-proteins@net.bio.net Sat Oct 04 23:00:00 1997 Path: biosci!OCELOT.RUTGERS.EDU!QUINONES From: QUINONES@OCELOT.RUTGERS.EDU (SUSAN QUINONES) Newsgroups: bionet.molbio.proteins Subject: Are there commercially available transcription factors? Date: 5 Oct 1997 10:47:06 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 12 Sender: daemon@net.bio.net Distribution: world Message-ID: <01IOG7BQYQC0920DS1@mbcl.rutgers.edu> NNTP-Posting-Host: net.bio.net X-URL: http://www.bio.net/hypermail/PROTEIN-ANALYSIS/ X-Mailer: Lynx, Version 2.6 X-Personal_name: Susan Quinones X-From: quinones@mbcl.rutgers.edu Does anyone know if any commercially available purified transcription factors OR vector system setups to generate specific transcription factors by in vitro translation that are suitable for GEL SHIFT studies? Thanks. From owner-proteins@net.bio.net Sat Oct 04 23:00:00 1997 Path: biosci!rutgers!nntp.upenn.edu!dsinc!spool.mu.edu!uwm.edu!vixen.cso.uiuc.edu!howland.erols.net!europa.clark.net!208.134.241.18!newsfeed.internetmci.com!192.48.96.126!in2.uu.net!news-nb.rutgers.edu!niflheim.rutgers.edu!not-for-mail From: meton@rci.rutgers.edu (Daniel Gonzalez) Newsgroups: bionet.molbio.proteins Subject: Re: Are there commercially available transcription factors? Date: 5 Oct 1997 19:33:24 -0400 Organization: Rutgers University Lines: 17 Message-ID: <619844$qal$1@niflheim.rutgers.edu> References: <01IOG7BQYQC0920DS1@mbcl.rutgers.edu> NNTP-Posting-Host: niflheim.rutgers.edu QUINONES@OCELOT.RUTGERS.EDU (SUSAN QUINONES) writes: >X-URL: http://www.bio.net/hypermail/PROTEIN-ANALYSIS/ >X-Mailer: Lynx, Version 2.6 >X-Personal_name: Susan Quinones >X-From: quinones@mbcl.rutgers.edu >Does anyone know if any commercially available purified transcription >factors OR vector system setups to generate specific transcription factors >by in vitro translation that are suitable for GEL SHIFT studies? >Thanks. You can try Santa Cruz Biotechnologies, they have a website; http://www.scbt.com/ you might want to try one of their proteins. Daniel Gonzalez From owner-proteins@net.bio.net Sun Oct 05 23:00:00 1997 Path: biosci!bloom-beacon.mit.edu!news-out.internetmci.com!newsfeed.internetmci.com!195.99.66.215!news-feed1.eu.concert.net!newsfeed.ecrc.net!192.174.65.44.MISMATCH!newscore.univie.ac.at!03-newsfeed.univie.ac.at!fstgal00.tu-graz.ac.at!balu.kfunigraz.ac.at!not-for-mail From: Markus Zettl Newsgroups: bionet.molbio.proteins Subject: solubilisation of membrane proteins Date: Mon, 06 Oct 1997 12:40:41 +0100 Organization: Karl-Franzens-Universitaet Graz Lines: 6 Message-ID: <3438CE39.7342B5EA@kfunigraz.ac.at> NNTP-Posting-Host: bimk14.kfunigraz.ac.at Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.01 [de] (Win95; I) X-Priority: 3 (Normal) I`m searching for a good method to solubilize a protein from membranes ? I`m looking for a method which gives me a high yield of membrane proteins ? Furthermore i`m interested in any source of information concerning this topic. Thanks for any help! From owner-proteins@net.bio.net Sun Oct 05 23:00:00 1997 Path: biosci!rutgers!uwm.edu!gsl-penn-ns.gsl.net!news-peer.gsl.net!news.gsl.net!gip.net!news.maxwell.syr.edu!news-was.dfn.de!news-fra1.dfn.de!news-ge.switch.ch!in2p3.fr!univ-lyon1.fr!cnusc.fr!ciril.fr!univ-angers.fr!univ-rennes1.fr!melon.univ-brest.fr!not-for-mail From: le Provost Olivier Newsgroups: bionet.molbio.proteins Subject: technical problem (cloning) Date: 6 Oct 1997 09:42:16 GMT Organization: Universite de Brest , France Lines: 6 Message-ID: <61abpo$p94$1@melon.univ-brest.fr> NNTP-Posting-Host: pc-lbcm-7.univ-ubs.fr We have a technical problem for a long time now. During cloning experiments in pBluescript, we always obtain recombinant plasmids lighter than pBluescript. None of them share an insert. The E.coli host strain used for these experiments is TG2. Usefull advices will be helpfull for us. Thanks From owner-proteins@net.bio.net Sun Oct 05 23:00:00 1997 Path: biosci!obgyn.ks.se!sstock From: sstock@obgyn.ks.se (Solveig Stock) Newsgroups: bionet.molbio.proteins Subject: Oxytocin receptor antibody Date: 6 Oct 1997 01:20:21 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 9 Sender: daemon@net.bio.net Distribution: world Message-ID: <3438ABFA.208C@obgyn.ks.se> NNTP-Posting-Host: net.bio.net Hello ! I would need an oxytocin receptor antibody, so if anyone know where I could find one I would be happy to know. Thanks Solveig S. From owner-proteins@net.bio.net Sun Oct 05 23:00:00 1997 Path: biosci!bloom-beacon.mit.edu!news-out.internetmci.com!newsfeed.internetmci.com!195.99.66.215!news-feed1.eu.concert.net!newsfeed.ecrc.net!192.174.65.44.MISMATCH!newscore.univie.ac.at!03-newsfeed.univie.ac.at!fstgal00.tu-graz.ac.at!balu.kfunigraz.ac.at!not-for-mail From: Markus Zettl Newsgroups: bionet.molbio.proteins Subject: solubilisation of membrane proteins Date: Mon, 06 Oct 1997 12:39:35 +0100 Organization: Karl-Franzens-Universitaet Graz Lines: 6 Message-ID: <3438CDF7.487E6678@kfunigraz.ac.at> NNTP-Posting-Host: bimk14.kfunigraz.ac.at Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.01 [de] (Win95; I) To: bionet.molbio.proteins.7tms_r@kfunigraz.ac.at X-Priority: 3 (Normal) I`m searching for a good method to solubilize a protein from membranes ? I`m looking for a method which gives me a high yield of membrane proteins ? Furthermore i`m interested in any source of information concerning this topic. Thanks for any help! From owner-proteins@net.bio.net Sun Oct 05 23:00:00 1997 Path: biosci!biosci!not-for-mail From: Gasior Newsgroups: bionet.molbio.proteins Subject: Chlorophyll Date: 6 Oct 1997 09:37:14 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 8 Sender: daemon@net.bio.net Distribution: world Message-ID: <01bcd16f$26d50140$LocalHost@biocom> NNTP-Posting-Host: net.bio.net I`m looking for a method to remove chlorophyll from solution containing integral protein complex (cytochrome b6f complex). Tomek W. gasior@ic.com.pl From owner-proteins@net.bio.net Sun Oct 05 23:00:00 1997 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.nacamar.de!uni-erlangen.de!winx03!wpxx02!not-for-mail From: krasel@wpxx02.toxi.uni-wuerzburg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins Subject: Re: solubilisation of membrane proteins Date: Mon, 6 Oct 1997 16:08:58 +0200 Organization: University of Wuerzburg, Germany Lines: 23 Message-ID: References: <3438CE39.7342B5EA@kfunigraz.ac.at> NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] Markus Zettl (markus.zettl@kfunigraz.ac.at) wrote: > I`m searching for a good method to solubilize a protein from membranes ? > I`m looking for a method which gives me a high yield of > membrane proteins ? If you have an integral membrane protein, you will have to play around with detergents. Unfortunately, there is no rule which detergent works best, therefore you will have to try which detergent best preserves the activity of your membrane protein. It also depends on what you want to do with the protein after solubilization. Calbiochem has a quite good brochure about the properties of various detergents. If you have a peripheral membrane protein, you might be lucky and get away with stuff like high salt stripping or alkaline washing. Good luck! --Cornelius. -- /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de SP4 */ /* "Science is the game we play with God to find out what His rules are." */ From owner-proteins@net.bio.net Sun Oct 05 23:00:00 1997 Path: biosci!agate!howland.erols.net!iagnet.net!newsfeed.acns.nwu.edu!news.cc.uic.edu!blackman From: blackman@tigger.cc.uic.edu (Samuel C. Blackman) Newsgroups: bionet.molbio.proteins,bionet.molbio.methds-reagnts` Subject: Protein Assay Choice (need assistance) Date: 6 Oct 1997 22:26:34 GMT Organization: University of Illinois at Chicago - ADN Computer Center Lines: 30 Message-ID: <61boiq$omk$1@piglet.cc.uic.edu> NNTP-Posting-Host: tigger.cc.uic.edu I have a thorny problem involving protein quantitation, and I was wondering whether or not there was an easy solution: The problem: - Proteins are solubilized in either 9M Urea/2-ME or 1%SDS/2-ME (making the resulting solutions incompatible with either Bradford or modified Lowry) - The microplate reader that I have access to only reads at 450, 490 or 650 nm (making the BCA assay not practical, since it likes to be read at 562 nm. The question: - Is there some protein assay that would allow me to work within the limitations state above, or do I need to TCA precipitate these proteins and swap buffers? Thanks! - Sam -- Samuel C. Blackman ! InterNet : blackman@tigger.uic.edu MD/PhD Student (5/8) ! Disclaimer: I speak for me, not UIC! Univ. of Ill. at Chicago ! Quote : "Quandro potro io finir di stupire?" Dept. of Pharmacology ! Phone : 312/996-4983 (lab) Fax: 312/996-1225 From owner-proteins@net.bio.net Sun Oct 05 23:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!206.229.87.25!news-peer.sprintlink.net!news.sprintlink.net!Sprint!uunet!in2.uu.net!hearst.acc.Virginia.EDU!murdoch.acc.Virginia.EDU!not-for-mail From: Rajesh Kumar Newsgroups: bionet.molbio.proteins Subject: Re: technical problem (cloning) Date: Mon, 06 Oct 1997 14:57:15 -0400 Organization: University of Virginia Lines: 6 Message-ID: <34393485.3C34@virginia.edu> References: <61abpo$p94$1@melon.univ-brest.fr> NNTP-Posting-Host: ppp-31-17.itc.virginia.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.02Gold (Macintosh; I; PPC) To: le Provost Olivier I guess your protein is toxic to bacteria. Try including 2% glucose in the media while growing the cells. This will help suppressing leaky expression as well as boost the growth. You may also try a different vector like pBR322. Hope that helps. Rajesh From owner-proteins@net.bio.net Sun Oct 05 23:00:00 1997 Path: biosci!BOTANY.UQ.EDU.AU!J.Marcus From: J.Marcus@BOTANY.UQ.EDU.AU ("Marcus, Dr J.") Newsgroups: bionet.molbio.proteins Subject: Re: Protein Assay Choice (need assistance) Date: 6 Oct 1997 23:23:51 -0700 Organization: Dept of Botany, Univ of Queensland Lines: 41 Sender: daemon@net.bio.net Distribution: world Message-ID: <5AB51E5608@botany.uq.edu.au> References: <61boiq$omk$1@piglet.cc.uic.edu> Reply-To: Marcus@tpp.uq.edu.au NNTP-Posting-Host: net.bio.net > I have a thorny problem involving protein quantitation, and I was > wondering whether or not there was an easy solution: > > The problem: > > - Proteins are solubilized in either 9M Urea/2-ME or 1%SDS/2-ME > (making the resulting solutions incompatible with either Bradford > or modified Lowry) > > - The microplate reader that I have access to only reads at 450, 490 > or 650 nm (making the BCA assay not practical, since it likes to > be read at 562 nm. > > The question: > > - Is there some protein assay that would allow me to work within > the limitations state above, or do I need to TCA precipitate > these proteins and swap buffers? > Although 650nm may not be optimal it should give you plenty of signal to measure protein concentration with. Your standard curve will tell you if the assay is working or not. All the best. John Marcus _________________________________________________________ John Marcus Marcus@tpp.uq.edu.au (Dr J.Marcus) Cooperative Research Centre for Tropical Plant Pathology 5th Level John Hines Building University of Queensland St. Lucia, QLD 4072 AUSTRALIA Fax: 61-7-3365-4771 Phone: 61-7-3365-4764 From owner-proteins@net.bio.net Mon Oct 06 23:00:00 1997 Path: biosci!BOTANY.UQ.EDU.AU!J.Marcus From: J.Marcus@BOTANY.UQ.EDU.AU ("Marcus, Dr J.") Newsgroups: bionet.molbio.proteins Subject: Re: Protein Assay Choice (need assistance) Date: 7 Oct 1997 17:28:02 -0700 Organization: Dept of Botany, Univ of Queensland Lines: 49 Sender: daemon@net.bio.net Distribution: world Message-ID: <6CC6002972@botany.uq.edu.au> References: Reply-To: Marcus@tpp.uq.edu.au NNTP-Posting-Host: net.bio.net Thanks John, I responded that BCA would not be a bad assay to use for the problem that was posted. I must admit I didn't even look at the solubilization buffer. Yes 2-ME is terrible for BCA when used at higher concentrations. I was only commenting on the wavelengths used for the assay. John Marcus John Berges wrote: > > > I have a thorny problem involving protein quantitation, and I was > > > wondering whether or not there was an easy solution: > > > > > > The problem: > > > > > > - Proteins are solubilized in either 9M Urea/2-ME or 1%SDS/2-ME > > > (making the resulting solutions incompatible with either Bradford > > > or modified Lowry) > > > > > > - The microplate reader that I have access to only reads at 450, 490 > > > or 650 nm (making the BCA assay not practical, since it likes to > > > be read at 562 nm. > > > > > > The question: > > > > > > - Is there some protein assay that would allow me to work within > > > the limitations state above, or do I need to TCA precipitate > > > these proteins and swap buffers? > > The BCA assay is very badly affected by any reducing agents > (DTT, mercaptoethanol)...I suspect TCA precipitation is > a good idea. Fully resolublizing the protein in the new > buffer will probably turn out to be the trick. _________________________________________________________ John Marcus Marcus@tpp.uq.edu.au (Dr J.Marcus) Cooperative Research Centre for Tropical Plant Pathology 5th Level John Hines Building University of Queensland St. Lucia, QLD 4072 AUSTRALIA Fax: 61-7-3365-4771 Phone: 61-7-3365-4764 From owner-proteins@net.bio.net Mon Oct 06 23:00:00 1997 Path: biosci!AMBER.BIOLOGY.GATECH.EDU!william From: william@AMBER.BIOLOGY.GATECH.EDU (William S. Hayes) Newsgroups: bionet.molbio.proteins Subject: UPDATED CONF NOTICE: Gene Discovery in silico Nov 6-9 1997 Date: 7 Oct 1997 15:39:53 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 209 Sender: daemon@net.bio.net Distribution: world Message-ID: <9710072238.AA23723@intron.biology.gatech.edu> NNTP-Posting-Host: net.bio.net GEORGIA TECH INTERNATIONAL CONFERENCE IN BIOINFORMATICS GENE DISCOVERY IN SILICO NOVEMBER 6 - 9, 1997 ATLANTA The focus of the conference is on gene identification and prediction of protein function using computer methods. SPONSORS: Georgia Tech College of Science Parker H. Petit Institute for Bioengineering and Bioscience at Georgia Tech SmithKline Beecham Gene Pro, Inc. Glaxo-Wellcome DATES: Early registration ends: October 9, 1997 Conference schedule: Registration opens at 6:00pm on Thursday, November 6, (with the reception at 8:00pm). The program starts 8:00am Friday, November 7 and ends at noon Sunday, November 9. LOCATION: The conference will be held at Renaissance Atlanta Hotel Downtown located near the center of 1996 Olympic development, close to Fox Theatre & Georgia Tech AGENDA: The conference agenda includes plenary talks (50 minutes for each talk and discussion) as well as poster session and round table discussion. WWW page now contain all titles as well as abstracts of the majority of the plenary talks. http://intron.biology.gatech.edu/~william/conference.html --------------------- Evening - Nov 6 Reception Morning - Nov 7 Peer Bork EMBL, Heidelberg & MDC, Berlin, Germany Predicting function from sequence Geoff Barton University of Oxford, Oxford, UK & EBI, Cambridge, UK Treading the benchmark tightrope: Making progress by measuring the accuracy of sequence analysis algorithms Jim Fickett SmithKline Beecham, King of Prussia, PA Analysis of Transcriptional Regulatory Regions Michael Zhang Cold Spring Harbor Lab, Long Island, NY On A New Strategy of Promoter Recognition Afternoon - Nov 7 Pavel Pevzner University of Southern California, Los-Angeles, CA The Twenty Questions Game with Genes Steven Salzberg Johns Hopkins University, Baltimore, MD Decision Trees and Markov Chains for Eukaryotic Gene Finding Steven Henikoff Fred Hutchinson Cancer Research Center, Seattle, WA Blocks-based methods for detecting homology and inferring function Stephen Altschul NCBI/NIH, Bethesda, MD More BLAST for Your Buck Late afternoon - Nov 7 (17:30 - 19:30) Poster Session Morning - Nov 8 Soren Brunak Technical University of Denmark, Copenhagen, Denmark DNA Structure and Sequence Periodicity in Human Promoters Sam Karlin Stanford University, Stanford, CA Codon biases and gene clusters Jean-Michel Claverie Struct & Genetic Information, CNRS, Marseille, France Self-identification of protein coding region in bacterial genomes Mark Borodovsky Georgia Institute of Technology, Atlanta, GA Three steps to complete gene identification in a new bacterial genome with GeneMark^3 Afternoon - Nov 8 Philipp Bucher ISREC, Lausanne, Switzerland Using generalized profiles for functional annotation of genome sequences Michael Gribskov San Diego Supercomputer Center, San Diego, CA Pattern Recognition Tools for Protein Sequences Gary Stormo University of Colorado, Boulder, CO Combining various types of evidence to predict gene structures Eugene Koonin NCBI/NIH, Bethesda, MD A genomic perspective on protein families Late afternoon - Nov 8 (17:30- 19:30) Round table discussion - "Annotating genomes Rapid first pass annotation of large scale sequences" Morning - Nov 9 Terry Gaasterland Argonne National Lab & Univ of Chicago, Chicago, IL Automated Analysis of Whole Genomes: Experiences and Lessons Anthony Kerlavage The Institute for Genomic Research, Rockville, MD Gene Finding and Functional Identification in Prokaryotic and Eukaryotic Genomes Roderic Guigo Inst Municipal d'Investigcio Medica, Barcelona, Spain Integrating multiple evidence to predict and annotate genes in genomic sequences Victor Solovyev Amgen, Inc., Thousand Oaks, CA & Sanger Centre, UK A new version of GeneFinder for analysis of genomic DNA with multiple genes STEERING & PROGRAM COMMITTEE: Mark Borodovsky, Co-Chair, Georgia Tech Soren Brunak Technical University of Denmark Jim Fickett SmithKline Beecham Eugene Koonin, Co-Chair, NCBI/NIH GEORGIA TECH ORGANIZING COMMITTEE: Chair Prof. Mark Borodovsky, School of Biology & Mathematics Advisory board Prof. Leonid Bunimovich, School of Mathematics Prof. Shamkant Navathe, College of Computing Poster session Dr. Alex Lukashin, School of Biology, Publicity William Hayes, School of Biology, william@intron.biology.gatech.edu Registration and general events Laura Olson, Department of Continuing Education, laura.olson@conted.gatech.edu Bonnie Griffin, School of Mathematics griffin@math.gatech.edu We thank Dr. Sorin Istrail and Dr. Ying Xu for help in compiling the e-mail distribution list. POSTER SUBMISSION To apply for participation in a poster session, please send the title and one page abstract of your poster, by October 3, to Dr. Alex Lukashin, by e-mail: lukashin@amber.biology.gatech.edu (preferred method) or by FAX: (404) 894-0519. You may send the abstract earlier. The acceptance of the high quality poster will be confirmed within five days upon submission. REGISTRATION Registration Fee: Early (until October 9) $295, Academic/government - $245 Late: $345, Academic/government - $295 The registration fee includes handout materials and meals provided by Renaissance hotel chefs for the whole conference time. Hotel Reservations: This conference will be held at the Renaissance Atlanta Hotel Downtown. Call Renaissance Atlanta Hotel 404-881-6000 for reservation with Georgia Tech room rate of $99/night by October 3, 1997. Please mention the Bioinformatics/Georgia Tech conference. Travel Discounts: Delta Air Lines offers special fares to attendees of Georgia Tech programs. Certain restrictions may apply. For information and reservations, call 1-800-241-6760 and refer to file U0175 (for domestic flights only). To obtain more information & the registration form, visit the WWW page: http://intron.biology.gatech.edu/~william/conference.html or contact us by e-mail: register@conted.swann.gatech.edu Phone: (404) 894-2400 Fax: (404) 894-8925 From owner-proteins@net.bio.net Mon Oct 06 23:00:00 1997 From: bogus1@hiv.edu.au (David Aldridge) Newsgroups: bionet.molbio.proteins Subject: Thiol reducing potential Date: Tue, 07 Oct 1997 23:14:04 +1000 Organization: MBCMR Lines: 15 Message-ID: NNTP-Posting-Host: 203.0.141.98 X-Newsreader: MT-NewsWatcher 2.3.3 Path: biosci!news.ic.sunysb.edu!news-pen-15.sprintlink.net!newsfeed.nysernet.net!news.nysernet.net!207.41.200.4!news-pen-4.sprintlink.net!206.229.87.26!news-east.sprintlink.net!news-dc-26.sprintlink.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news-xfer.netaxs.com!news.misty.com!news.uwa.edu.au!munnari.OZ.AU!cs.mu.OZ.AU!news.unimelb.edu.au!bogus1 Could somebody point me to a reference which lists the relative reducing potentials of various thiols. I'm particularly interested in GSH relative to DTT. Thanks in advance -- David Aldridge ____________________________________________________________________, Research Officer, |Domain Administrator, | Macfarlane Burnet Centre |Australian National Centre in HIV | for Medical Research. |Virology Research. | http://www.burnet.edu.au |http://www.hiv.edu.au | --------------------------------------------------------------------* The reply address above will only be valid until spammed. From owner-proteins@net.bio.net Mon Oct 06 23:00:00 1997 Path: biosci!agate!newsfeed.kornet.nm.kr!howland.erols.net!surfnet.nl!rl0001.unimaas.nl!fys0210uns50.unimaas.nl!Michael.Vork From: Michael.Vork@FYS.rulimburg.nl (Michael Vork) Newsgroups: bionet.molbio.proteins Subject: Immobilize proteins on nitrocellulose with UV crosslinking Date: Tue, 7 Oct 1997 09:29:07 GMT Organization: Rijksuniversiteit Limburg Lines: 8 Message-ID: NNTP-Posting-Host: fys0210uns50.unimaas.nl X-Newsreader: Trumpet for Windows [Version 1.0 Rev A] Has anybody ever tried crosslinking proteins on nitrocellulose using UV. If so, please let me know what you think about it and if you can recommend it. Thanx, Michael Vork From owner-proteins@net.bio.net Mon Oct 06 23:00:00 1997 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsm.ibm.net!ibm.net!news.biu.ac.il!news.huji.ac.il!not-for-mail From: "Jonathan B. Marder" Newsgroups: bionet.molbio.proteins Subject: Re: Chlorophyll Date: Tue, 7 Oct 1997 08:42:52 +0200 Organization: Hebrew University Lines: 21 Message-ID: <61cllg$p84$1@news.huji.ac.il> References: <01bcd16f$26d50140$LocalHost@biocom> NNTP-Posting-Host: marder.agri.huji.ac.il Mime-Version: 1.0 Content-Type: text/plain; charset="utf-7" Content-Transfer-Encoding: 7bit X-Newsreader: Microsoft Outlook Express 4.71.1712.3 X-MimeOLE: Produced By Microsoft MimeOLE V4.71.1712.3 Gasior wrote in message +ADw-01bcd16f+ACQ-26d50140+ACQ-LocalHost+AEA-biocom+AD4-... +AD4-I+AGA-m looking for a method to remove chlorophyll from solution containing +AD4-integral protein complex (cytochrome b6f complex). +AD4- +AD4-Tomek W. +AD4- +AD4-gasior+AEA-ic.com.pl +AD4- Actually, there is a claim that the b6f complex may actually bind some chlorophyll. You should have a look at a recent abstract by Barbato et al. in Plant Physiology and Biochemistry, Special Issue, 1996 - that's the issue with abstracts for the Florence FESPP congress. Jonathan B. Marder +ADw-MARDER+AEA-agri.huji.ac.il+AD4- Department of Agricultural Botany, The Hebrew University of Jerusalem Faculty of Agriculture, P.O.Box 12, Rehovot 76100, ISRAEL Phone: +-972 8 9481918 Fax: +-972 8 9467763 Web page: http://www.agri.huji.ac.il/+AH4-marder From owner-proteins@net.bio.net Mon Oct 06 23:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!141.211.144.13!newsxfer3.itd.umich.edu!oleane!pasteur.fr!infobiogen.fr!lovelace.infobiogen.fr!ahelme From: Alix Helme-Guizon Newsgroups: bionet.molbio.proteins Subject: olfactory bulb synaptosomes protocol Date: Tue, 7 Oct 1997 17:53:34 +0200 Organization: "GIS INFOBIOGEN, 7 rue Guy Moquet BP8, 94801 VILLEJUIF, France" Lines: 7 Message-ID: NNTP-Posting-Host: lovelace.infobiogen.fr Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII hi i'm looking for a protocol to prepare synaptosomes from an olfactory bulb. the synaptosomes don't need to be intact as they will only be used for western blotting thanks for your help alix From owner-proteins@net.bio.net Mon Oct 06 23:00:00 1997 Path: biosci!agate!newsfeed.kornet.nm.kr!newsfeed.dacom.co.kr!newsfeed.direct.ca!news-feed1.tiac.net!news-master.tiac.net!news@tiac.net From: jamesl@healthtech.com (James W. Larkin) Newsgroups: bionet.molbio.proteins Subject: 2nd Annual Japanese COMBINATORIAL CHEMISTRY & HIGH THROUGHPUT SCREENING Date: 7 Oct 1997 15:23:23 GMT Organization: Cambridge Healthtech Institute Lines: 266 Message-ID: <61dk5b$cqm@news-central.tiac.net> NNTP-Posting-Host: 207.60.238.8 Mime-Version: 1.0 Content-Type: Text/Plain; charset=ISO-8859-1 X-Newsreader: WinVN 0.99.9 Beta 1 (x86 32bit) 2nd Annual Japanese COMBINATORIAL CHEMISTRY & HIGH THROUGHPUT SCREENING October 29-31, 1997 Keio Plaza Inter-Continental,Tokyo, Japan Corporate Sponsor: Tripos Corp. Corporate Support: Trega Biosciences Oxford Asymmetry Arqule, Inc On Wednesday, October 29, there will be a series of Corporate Partnering presentations. Material from each of the presenting companies will be posted on the Cambridge Healthtech Web page, to allow for review by attendees prior to the meeting. Invitations will be extended to a number of pharmaceutical executives throughout Japan and elsewhere in Asia to attend this portion of the meeting on a complimentary basis. At the end of the partnering session, the exhibits will be available for viewing as well. Initial Partnering Presentations Arris Pharmaceuticals Oxford Asymmetry Aurora Biosciences Trega Biosciences IRORI Quantum Microchemistry Tripos Corporation Oncogene Science, Inc. Versicor Orchid BioComputer, Inc. LIBRARIES APPLICATIONS OF COMBINATORIAL CHEMISTRY TO DRUG DISCOVERY IN INFECTIOUS DISEASE Dr. Eric M. Gordon, Versicor LOW-DIVERSITY SMART LIBRARYTM TECHNOLOGY: LIBRARIES FOR THE DEVELOPMENT OF SMALL MOLECULE Dr. Michael Kahn, Molecumetics, Ltd. THE DESIGN OF A UNIVERSAL INFORMER LIBRARY FOR DRUG DISCOVERY Dr. Peter Myers, CombiChem, Inc. COMBINATORIAL CHEMISTRY: FROM CONCEPT TO APPLICATION AS A DRUG DISCOVERY TOOL Dr. Alex Polinsky, Alanex Corporation COMBINATORIAL ORGANIC SYNTHESIS USING RADIO FREQUENCY MEMORY DEVICES Dr. Anthony Czarnik, IRORI Quantum Microchemistry MICROFABRICATION FOR MICROCHEMISTRY IN HIGH-THROUGHPUT DRUG DISCOVERY Dr. Pat Conway, Orchid BioComputer, Inc. TITLE TO BE ANNOUNCED Dr. David Casebier, ArQule, Inc. COMBINATORIAL SYNTHESIS DEVELOPMENT OF SYNTHETIC TOOLS FOR PREPARATION OF SMALL MOLECULE LIBRARIES Dr. Tony Baxter, Oxford Asymmetry ADVANCES IN METHODOLOGY FOR HIGH-THROUGHPUT SOLUTION AND SOLID-PHASE SYNTHESIS OF SMALL MOLECULES Dr. Douglas Livingston, Arris Pharmaceutical Corporation SYNTHESIS OF HETEROCYCLES ON THE SOLID PHASE Dr. Michael J. Green, Trega Biosciences TITLE TO BE ANNOUNCED Dr. Alasdair MacDonald, Argonaut Technologies, Inc. ROBOTICS AND SMALL-SIZED CHEMISTRY FOR COMBINATORIAL REACTION AUTOMATION Dr. Akito Tanaka, Fujisawa Pharmaceuticals (tentative) TITLE TO BE ANNOUNCED Advanced ChemTech (invited) INFORMATICS OPTIVERSE™ AND CHEMSPACE™: AN INTEGRATED STRATEGY FOR DESIGN AND SYNTHESIS OF LEAD DISCOVERY AND LEAD REFINEMENT LIBRARIES USING A NOVEL VIRTUAL LIBRARY TECHNOLOGY Dr. Peter Hecht, Tripos GmbH, Munich TITLE TO BE ANNOUNCED Dr. Barry Robson, MDL Information Systems (invited) NOVEL SOFTWARE TOOLS FOR CHEMICAL DIVERSITY AND COMBINATORIAL CHEMISTRY Dr. Robert Pearlman, University of Texas, Austin SCREENING AUTOMATED HTS AT AMGEN: AN OVERVIEW OF WORKSTATIONS AND INTEGRATED SYSTEMS APPROACHES Dr. Jason Armstrong, Amgen NOVEL FLUOROGENIC BIOASSAY TECHNOLOGY FOR RAPID SCREEN DEVELOPMENT FOR ULTRA HIGH-THROUGHPUT SCREENING Dr. Harry Stylli, Aurora Biosciences STRATEGIC LEVERAGING OF AN ESTABLISHED DRUG DISCOVERY ENGINE Dr. Mel Reichman, Oncogene Science, Inc. TITLE TO BE ANNOUNCED Dr. Shina Tanaka, Nihon Millipore (tentative) TITLE TO BE ANNOUNCED Dr. Kurt Schilling, Molecular Devices (invited) Cambridge Healthtech is pleased to again work closely with the Japan Combinatorial Chemistry Focus Group (J.C.C.F.) for this second annual meeting. Prior to the opening of the technical meeting there will be a tutorial workshop, offered in Japanese, for review of technical terms and concepts that will be covered during the meeting. There will also be a series of Corporate Partnering Presentations, to which leading decision makers from Asian pharmaceutical companies will be invited. The technical program will first focus on develop-ments in the design and synthesis of small molecule combinatorial libraries for lead identification and lead optimization. Advances in high throughput screening, including automation, novel assays, data handling, and interpretation, as well as integration of these issues, will then be featured. Thirty exhibitors participated in the trade exhibit last year, and more are expected to take advantage of this unique opportunity to be in touch with key researchers and managers attending this meeting. Interest and application of these two key technologies are expected to follow a similar pattern of rapid growth as is being experienced elsewhere throughout the world. INITIAL EXHIBITORS AMERSHAM KK NALGE NUNC ARGONAUT TECHNOLOGIES NIHON MILLIPORE BECKMAN INSTRUMENTS OXFORD ASYMMETRY (JAPAN) LTD. ROBBINS SCIENTIFIC CEREP TECAN CO. CONTACT SERVICE CO. TEIJIN SYSTEMS CORNING COSTAR CORP. TOYOBO DAIICHI PURE CHEMICALS TRIPOS CORP. M & S INSTRUMENTS WHATMAN POLYFILTRONICS MATRIX TECHNOLOGIES CALL FOR EXHIBITORS Space is available for organizations interested in exhibiting technologyor services to reach the targeted audience that will be attending this meeting on Combinatorial Chemistry & High Throughput Screening. Please contact Jim MacNeil of Cambridge Healthtech Institute at 617-630-1341 to obtain an exhibitor package or to inquire about offering a workshop during the meeting. Exhibit space is limited so call now to reserve a space at this premier event. HOTEL INFORMATION Keio Plaza Inter-Continental Room reservations must be made 2-1, Nishi-Shinjuku 2-Chome through Cambridge Healthtech Shinjuku-ku, Tokyo 160 JAPAN Institute (CHI). You must fill out the reservation form and send it T: 81-3-3344-0111 back to CHI by Sept. 12, 1997 F: 81-3-3345-8269 (reservation forms may not be accepted after this date and will Cut-off Date:September 12, 1997 be subject to hotel reservation policies). Limited rooms are a Room Rate: 18,000 JPY-Single vailable and are subject to 20,000 JPY-Double availability. Please call CHI with any questions, e-mail Jennifer Katz at jkatz@healthtech.com, or contact us by fax at 617-630-1325. TRAVEL INFORMATION TRAVELWORLD T: 717-288-9311 or 800-828-6033 601 Market Street F: 717-288-4693 Kingston, PA 18704 Exclusive airline discounts are available on specific airlines when tickets are purchased through Travelworld at least 14 days prior to the meeting date. Some restrictions apply. Handicapped Equal Access: In accordance with the ADA, Cambridge Healthtech Institute is pleased to arrange for special accommodations for attendees with special needs. All request for such assistance must be submitted in writing to CHI at least 30 days prior to the start of the meeting. Each registration includes all conference sessions, posters and exhibits, one luncheon and reception, continental breakfasts, all refreshment breaks, and a copy of the document binder. A group rate is available for three or more people from the same organization. Call for more details. SUBSTITUTION CANCELLATION POLICY In the event that you need to cancel a registration you may: Transfer your registration to a colleague within your organization. Credit your registration to another Cambridge Healthtech Institute program. Request a refund minus a $75 processing fee. Request a refund minus the cost ($195) of ordering a copy of the document binder. Cancellations will only be accepted up to one week prior to the conference. Program and speakers are subject to change. --------------------CUT AND PRINT HERE----------------------------- YES |__| Please register me for: E-Mail #568E-J70 Combinatorial Chemistry & High Throughput Screening On-site or Late Registration (after September 12, 1997) $995 |__| Commercial $495 |__| Academic, Government, Hospital-Affiliated |__| Complimentary Registration for Partnering Presentations ONLY FIRST NAME:______________________________________________________ LAST NAME:_______________________________________________________ TITLE:___________________________________________________________ DIV./DEPT.:______________________________________________________ COMPANY:_________________________________________________________ ADDRESS:_________________________________________________________ City/State/ZIP:__________________________________________________ COUNTRY:_________________________________________________________ TELEPHONE:____________________________ Fax:______________________ E-MAIL:__________________________________________________________ |__| Please send information on exhibiting and opportunities to present workshops. |__| Enclosed is a check or money order payable to Cambridge Healthtech Institute, drawn on a U.S. bank, in U.S. currency. |__| Please charge: |__| AMEX (15 digits) |__| Visa (13 to 16 digits) |__| MasterCard (16 digits) Card #:___________________________________________________________ Exp. Date:________________________________________________________ Cardholder's Name:________________________________________________ Signature:________________________________________________________ Cardholder's Address (if different from above):___________________ __________________________________________________________________ |__| Reserve with credit card information listed above and invoice me. (Invoices must be paid in full by the deadline to retain registration discount. Invoices unpaid one week prior to conference will be billed to credit card at full registration rate.) If you plan to register on site, please check with CHI beforehand for space availability. KEIO PLAZA INTER-CONTINENTAL RESERVATION FORM (only valid until September 12, 1997) * SIGNATURE:_______________________________________________________ (Authorizes agreement to the terms under Hotel Information; please note your reservation form will not be processed without signature.) Hotel arrival date:_____ / _____ Hotel departure date:_____ / ___ Month / Day Month / Day Special Requests (subject to availability):________________________ ___________________________________________________________________ |__| Credit Card to Guarantee Reservation: |__| AMEX (15 digits) |__| Visa (13 to 16 digits) |__| MasterCard (16 digits) |__| Use above credit card information. Reservations WILL NOT be accepted without a credit card guarantee. CARD #:_____________________________________ EXP. DATE:_________________ CARDHOLDER’S NAME: _________________________ SIGNATURE:_________________ FAX or MAIL your reservation/registration to: Cambridge Healthtech Institute 1037 Chestnut Street Newton Upper Falls, MA 02164 tel: 617-630-1300 fax: 617-630-1325 e-mail: chi@healthtech.com http:www.healthtech.com/conferences/ From owner-proteins@net.bio.net Mon Oct 06 23:00:00 1997 Path: biosci!agate!howland.erols.net!news-peer.gsl.net!news.gsl.net!gip.net!fu-berlin.de!derma0001.ukbf.fu-berlin.DE!not-for-mail From: Oliver Politz Newsgroups: bionet.microbiology,bionet.molbio.methds-reagnts,bionet.molbio.proteins Subject: antibody towards dnaB E.coli wanted Date: Tue, 07 Oct 1997 16:14:52 +0100 Organization: Freie Universitaet Berlin Lines: 8 Message-ID: <343A51EB.F2161D10@medizin.fu-berlin.de> NNTP-Posting-Host: derma0001.ukbf.fu-berlin.de (160.45.173.203) Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Access: 16 17 19 X-Mailer: Mozilla 4.01 [en] (WinNT; I) X-Priority: 3 (Normal) Xref: biosci bionet.microbiology:11235 bionet.molbio.methds-reagnts:61797 bionet.molbio.proteins:11613 Hello, I am looking for an antibody towards dnaB of E.coli or similar proteins (i.e. dnaC Bacillus subtilis) for western blot detection of a potential homologue gene. If anybody knows where to get it or can send me a small sample it would be a great help to finish my project. Thanks Oliver From owner-proteins@net.bio.net Mon Oct 06 23:00:00 1997 Path: biosci!daresbury!lyra.csx.cam.ac.uk!server1.netnews.ja.net!warwick!leicester!usenet From: "Dr E. Buxbaum" Newsgroups: bionet.molbio.proteins Subject: Re: Immobilize proteins on nitrocellulose with UV crosslinking Date: Tue, 07 Oct 1997 18:33:58 -0700 Organization: University of Leicester (PCFS User) Lines: 11 Message-ID: <343AE306.5CC9@le.ac.uk> References: NNTP-Posting-Host: pc47.msb.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Win16; I) Michael Vork wrote: > > Has anybody ever tried crosslinking proteins on nitrocellulose using UV. If > so, please let me know what you think about it and if you can recommend it. What do you widh to do with those proteins? Normally you'l be better of binding them to PVDF membranes (by electro- or dot-blotting, filtration or incubation of membranes with protein solution) instead of NC. Cross-linking is then no longer required even under harsh conditions (like gas phase sequencing). The procedure is also mild enough to maintain the binding and catalytic properties of enzymes. From owner-proteins@net.bio.net Tue Oct 07 23:00:00 1997 Path: biosci!bloom-beacon.mit.edu!thetimes.pixel.kodak.com!news.kodak.com!news-pen-16.sprintlink.net!newsfeed.nysernet.net!news.nysernet.net!207.41.200.4!news-pen-4.sprintlink.net!206.229.87.26!news-east.sprintlink.net!news-dc-26.sprintlink.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!ix.netcom.com!news From: Michael Salvucci Newsgroups: bionet.molbio.proteins Subject: HIC of Membrane Protein Date: Wed, 08 Oct 1997 20:55:11 GMT Organization: Netcom Lines: 9 Message-ID: <61gs6n$1al@dfw-ixnews6.ix.netcom.com> NNTP-Posting-Host: phn-az9-23.ix.netcom.com X-NETCOM-Date: Wed Oct 08 3:59:03 PM CDT 1997 X-Newsreader: NETCOMplete/3.27 To Members of the Protein Community, Does anyone have any experience separating membrane proteins using Hydrophobic Interaction Chromatography? The protein of interest is soluble and active in non-ionic detergents. I would appreciate any information on conditions and columns, and any general recommendations. NB, I would like to avoid reverse phase HPLC since I need to preserve activity. Thanks.........MIKE From owner-proteins@net.bio.net Tue Oct 07 23:00:00 1997 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeed.internetmci.com!203.12.176.153!news.mel.aone.net.au!newsfeed-in.aone.net.au!news.mel.connect.com.au!munnari.OZ.AU!bunyip.cc.uq.edu.au!not-for-mail From: "nH" Newsgroups: bionet.molbio.proteins Subject: REfolding and the drama that is Date: 9 Oct 1997 04:38:44 GMT Organization: University of Queensland Lines: 5 Message-ID: <01bcd46d$38d8fe40$1f6f6682@default> NNTP-Posting-Host: mdnhussa.slip.cc.uq.edu.au X-Newsreader: Microsoft Internet News 4.70.1155 Hello is there any info available on line for refolding proteins in inclusion bodies in bacteria ? I'm astudent..hence lack of knowledge and drive to look it up at the library.. From owner-proteins@net.bio.net Tue Oct 07 23:00:00 1997 Path: biosci!agate!howland.erols.net!feed1.news.erols.com!eru.mt.luth.se!luth.se!news.lth.se!not-for-mail From: David Hagerberg Newsgroups: bionet.molbio.proteins Subject: Antibacterial agent Date: Wed, 08 Oct 1997 14:38:46 +0100 Organization: Dep Microbial Ecology, Lund University Lines: 14 Message-ID: <343B8CE6.526D@mbioekol.lu.se> NNTP-Posting-Host: pc2.ekol.lu.se Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (Win95; I) Hello! My brother-in-law has been recommended to use sodium azide (NaN3) as an antibacterial agent in his antibody-storage-medium. What concentration should he use? Azide is rather nasty, could he use anything else? The answers should be sent to Henrik.Mosen@farm.lu.se Best Wishes! From owner-proteins@net.bio.net Tue Oct 07 23:00:00 1997 Path: biosci!agate!howland.erols.net!newsfeed.internetmci.com!138.95.18.2!wilbur.sequent.com!newsfeed.orst.edu!129.101.119.221.MISMATCH!news.uidaho.edu!lss34.campus.uidaho.edu!user From: cweil@uidaho.edu (Clifford Weil) Newsgroups: bionet.molbio.proteins Subject: DIRECTOR OF MOLECULAR BIOLOGY TRAINING FACILITY Date: Wed, 08 Oct 1997 17:37:25 -0700 Organization: University of Idaho, Dept. of Biological Sciences Lines: 15 Distribution: bionet Message-ID: NNTP-Posting-Host: lss34.campus.uidaho.edu Mime-Version: 1.0 Content-Type: text/plain; charset=US-ASCII Content-Transfer-Encoding: 7bit DIRECTOR OF MOLECULAR BIOLOGY TRAINING FACILITY The University of Idaho Biological Sciences Dept. seeks a Ph.D. to head a fully equipped laboratory for training students and faculty in a broad range of molecular techniques (DNA, RNA and proteins). At least two years of postdoctoral experience and demonstrated skill in training others required; experience with phosphorimaging and plant/animal cell culture desirable. Potential for independent or collaborative research and grants. Send letter of application, CV, description of research and techniques experience and names, addresses and telephone numbers of three references by November 15, 1997 to: Mol. Biol. Director Search, Dept. of Biological Sciences, University of Idaho, Moscow, ID 83844-3051. The University of Idaho is an equal opportunity/ affirmative action employer. From owner-proteins@net.bio.net Tue Oct 07 23:00:00 1997 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!newsfeeds.sol.net!uwm.edu!newsspool.doit.wisc.edu!news.itis.com!news.doit.wisc.edu!default From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: HIC of Membrane Protein Date: Thu, 09 Oct 1997 00:52:25 GMT Organization: UW-Madison Lines: 20 Message-ID: <61h9s9$3dc_002@doit.wisc.edu> References: <61gs6n$1al@dfw-ixnews6.ix.netcom.com> NNTP-Posting-Host: f182-145.net.wisc.edu X-No-Archive: Yes In article <61gs6n$1al@dfw-ixnews6.ix.netcom.com>, Michael Salvucci wrote: #To Members of the Protein Community, # #Does anyone have any experience separating membrane proteins using Hydrophobic #Interaction Chromatography? The protein of interest is soluble and active in #non-ionic detergents. I would appreciate any information on conditions and #columns, and any general recommendations. NB, I would like to avoid reverse #phase HPLC since I need to preserve activity. As far as chromatography is concerned, no general recommendation can generally be made :-) In my experience, for large proteins yeilds tend to be miserably low making the whole thing meaningless. Otherwise, with membrane proteins everything is the same as with truly soluble one, the only difference is that the everything is done but in the presence of detergents. Trick for phenyl-based matrixes: just changing detergent from tween to triton may elute significant portion of bound protein (triton's ring plays some role?). Dima From owner-proteins@net.bio.net Wed Oct 08 23:00:00 1997 Path: biosci!uni-konstanz.de!Robert.Deicher From: Robert.Deicher@uni-konstanz.de (Robert Deicher) Newsgroups: bionet.molbio.proteins Subject: re: preventing protein degradation Date: 9 Oct 1997 11:12:06 -0700 Organization: University of Konstanz Lines: 17 Sender: daemon@net.bio.net Distribution: world Message-ID: <343D1DD3.4837@uni-konstanz.de> Reply-To: Robert.Deicher@uni-konstanz.de NNTP-Posting-Host: net.bio.net You could try the following: 1. Protein degradation may well be dependent on Sf9 cell lysis by the baculovirus. When doing a time-course of your culture you should see a growing number of degradation products with time. You should be able to determine the optimal time to harvest, i.e. good protein yield and only minor degradation products. The best time point to harvest is probably just before virus-induced cell lysis. 2. You should try to add proteinase inhibitors directly to the cell culture, beginning for instance with day 2 (to minimize costs). I tried that with Pefabloc and the Complete EDTA free Proteinase Inhibitor cocktail sold by Boehringer Mannheim. The latter worked (at a forth of the concentration indicated by Boehringer Mannheim), the former did work not very well at 200 5M/l. Robert.Deicher@uni-konstanz.de From owner-proteins@net.bio.net Wed Oct 08 23:00:00 1997 Path: biosci!daresbury!uninett.no!news.algonet.se!news.maxwell.syr.edu!news-peer.sprintlink.net!news.sprintlink.net!Sprint!uunet!in5.uu.net!ozemail!news.mel.aone.net.au!newsfeed-in.aone.net.au!inferno.mpx.com.au!news.unimelb.edu.au!ludwignt-4 From: murphy_r@licre.ludwig.edu.au (Roger Murphy) Newsgroups: bionet.molbio.proteins Subject: bionet.molbio.proteins Date: Fri, 10 Oct 97 04:06:56 GMT Organization: Ludwig Institute Lines: 22 Message-ID: <61kcr9$3oj$1@izvestia.its.unimelb.edu.au> NNTP-Posting-Host: ludwignt-4.austin.unimelb.edu.au X-Newsreader: News Xpress 2.0 Beta #0 Keyword: virus chromatography Has anyone out there come across a reasoanble review of how viruses behave on chromatography systems? We are producing IgG's from transfected cells for clinical studies and need to satisfy the FDA about viral clearance. It would help us design protein purification protocols if we new how virus particles behave on things like Q- and SPp Sepharose. All comments/suggestions gratefully received Roger Murphy Dr. Roger Murphy Biological Production Facility Ludwig Institute for Cancer Research Austin & Repatriation Medical Centre Studley Road, Heidelberg, Vic. 3084 Tel 61-3-94965463 Fax 61-3-94965436 Email murphy_r@licre.ludwig.edu.au From owner-proteins@net.bio.net Wed Oct 08 23:00:00 1997 Path: biosci!rutgers!nntp.upenn.edu!dsinc!spool.mu.edu!uwm.edu!vixen.cso.uiuc.edu!iagnet.net!newsfeed.internetmci.com!207.69.200.61!mindspring!news.mindspring.com!usenet From: Rick Bright Newsgroups: bionet.molbio.proteins Subject: Efficient Protein Transfer Date: Thu, 09 Oct 1997 23:52:07 -0400 Organization: Emory University, Molecular Therapeutics & Toxicology Lines: 19 Message-ID: <343DA667.ECE8013E@emory.edu> NNTP-Posting-Host: user-37kb6gr.dialup.mindspring.com Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Server-Date: 10 Oct 1997 03:52:58 GMT X-Mailer: Mozilla 4.03 [en] (Win95; U) I am having trouble transferring enough myosin out of my 7.5% SDS-PAGE, onto a Nylon membrane. I am using Tris-Glycine/10% MeOH transfer buffer, equilibrating gel in same, activating membrane in 100% MeOH. I have tried 3 hr transfer 250mA and overnight transfer 150mA. I am staining the gel with Coumassie after transfer and have very large myosin bands and no other bands. Can anyone suggest better transfer conditions, buffers, gel concentration, etc? Since I an using nylon, I don't think I need the MeOH in the transfer buffer which may help, and I may drop down to 5.5% gel. Do you think either variation would improve the transfer? Thanks, Rick Bright P.S. I am actually probing with F1652 (fetal myosin) after transfer, but it is the same size as myosin. From owner-proteins@net.bio.net Wed Oct 08 23:00:00 1997 Path: biosci!rutgers!nntp.upenn.edu!news.misty.com!www.nntp.primenet.com!globalcenter1!news.primenet.com!nntp.primenet.com!newsfeed.nacamar.de!newscore.univie.ac.at!03-newsfeed.univie.ac.at!fstgal00.tu-graz.ac.at!balu.kfunigraz.ac.at!not-for-mail From: Markus Zettl Newsgroups: bionet.molbio.proteins Subject: break softly Proteininteractions Date: Thu, 09 Oct 1997 18:52:36 +0100 Organization: Karl-Franzens-Universitaet Graz Lines: 3 Message-ID: <343D19E4.2F505718@kfunigraz.ac.at> NNTP-Posting-Host: bimk12.kfunigraz.ac.at Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: quoted-printable X-Mailer: Mozilla 4.01 [de] (Win95; I) X-Priority: 3 (Normal) How can softly break protein-protein interactions? I=B4m working with a kind of affinitychromatography. After seperation Iwould like to detect the protein by SDS-page. From owner-proteins@net.bio.net Wed Oct 08 23:00:00 1997 Path: biosci!CNAS.SMSU.EDU!dac012f From: dac012f@CNAS.SMSU.EDU (Dean Cuebas) Newsgroups: bionet.molbio.proteins Subject: Protein Purification Systems Date: 9 Oct 1997 09:06:12 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 38 Sender: daemon@net.bio.net Distribution: world Message-ID: <199710091605.LAA27998@cnas.smsu.edu> NNTP-Posting-Host: net.bio.net I am in the market for a protein purification system and I had demonstrations for the Perseptive Sprint system and the comparable AKTA system, and felt that the AKTA software was far superior, but the replication of results (actual performance) on the Perseptive system was far superior. Interestingly, I noticed a bug in the Perseptive software (the scrolling chart recorder occasionally did not update, even though it was set for real time, unless the window was expanded to full size.) Has anyone else seen this? The sales rep says they can't reproduce it back in their labs.) As far as reproducibility, repeat sample injections were right on the money. So my gut feeling, is that the Perseptive system actually performs better, but the AKTA software is much more comprehensive. Any feedback would be very helpful. These systems are not cheap, and any feedback from people who own either of these systems will be greatly appreciated. Thanks millions in advance, Dr. Dean Cuebas ////////////////////////////////////////////////////////////////////// \ Dean Cuebas, PhD., Dept. of Chemistry / \ Southwest Missouri State University / \ Springfield, Missouri 65804 http://science.smsu.edu/chemistry / \ FAX/Ph (417)-836-8567 email: dac012f@cnas.smsu.edu / \ We are what we repeatedly do. / \ Excellence, then, is not an act but a habit. -Aristotle / \ Analytical Biochem. Lipid Metabolism Enzyme Assays / \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ From owner-proteins@net.bio.net Wed Oct 08 23:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!europa.clark.net!205.252.116.205!howland.erols.net!news.starnet.net!news.starnet.net!newsreader.wustl.edu!not-for-mail From: "George W. Chacko" Newsgroups: bionet.molbio.proteins Subject: Chemical Stability of Phosphotyrosine Date: Thu, 09 Oct 1997 14:18:05 +0000 Organization: Washington University in St. Louis Lines: 18 Message-ID: <343CE79D.190D@im.wustl.edu> Reply-To: gchacko@im.wustl.edu NNTP-Posting-Host: 128.252.178.173 Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.0Gold (Macintosh; I; PPC) Could someone point me at a good reference source for questions about the chemical stability of the phosphate group on phosphorylated proteins (specifically on tyrosine). I am most interested in the effect of pH, solvents and temperature. Yes, I know that phosphorylated tyrosine, unlike phosphorylated serine and threonine, is relatively stable to treatment with 1M KOH at 55C for 2 hours. Responses by e-mail to gchacko@im.wustl.edu would be highly appreciated. Thanks GC George W. Chacko Washington University School of Medicine 660 S. Euclid Avenue St. Louis, MO 63110 From owner-proteins@net.bio.net Wed Oct 08 23:00:00 1997 Path: biosci!uni-konstanz.de!Robert.Deicher From: Robert.Deicher@uni-konstanz.de (Robert Deicher) Newsgroups: bionet.molbio.proteins Subject: Native protein purification Date: 9 Oct 1997 11:24:29 -0700 Organization: University of Konstanz Lines: 6 Sender: daemon@net.bio.net Distribution: world Message-ID: <343D20BE.669E@uni-konstanz.de> Reply-To: Robert.Deicher@uni-konstanz.de NNTP-Posting-Host: net.bio.net I am trying to purify a 6xHis tagged multi zinc finger protein from baculovirus infected Sf9 cells under native conditions via Ni-NTA, however the protein yield is extremely low. Any suggestions for pushing up the protein yield? Should I first purify under denaturing conditions and than try to refold? Thanks for any help, Robert.Deicher@uni-konstanz.de From owner-proteins@net.bio.net Wed Oct 08 23:00:00 1997 Path: biosci!daresbury!uninett.no!sn.no!news-feed.ifi.uio.no!recycled.news.erols.com!howland.erols.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!uunet!in4.uu.net!munnari.OZ.AU!news.unimelb.edu.au!not-for-mail From: Roger Murphy Newsgroups: bionet.molbio.proteins Subject: 3rd Australian Peptide Conference Date: Fri, 10 Oct 1997 15:49:00 +1000 Organization: Ludwig Institute Lines: 27 Message-ID: <343DC1CC.1A84@licre.ludwig.edu.au> NNTP-Posting-Host: ludwignt-4.austin.unimelb.edu.au Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (WinNT; I) Planning is well under way for the 3rd. Australian Peptide Conference, to be held at the Laguna Keys resort, Whitsunday Islands, in tropical Queensland, from 4-9 October, 1998. The committee has established a web site which is currently under construction. I would urge all of you to have a look at this web site and bookmark it so you can keep abreast of developments. Registration information should be available early in 1998. http://www.hfi.unimelb.edu.au/peptideoz Hope to see as many of you there as possible. Roger Murphy Dr. Roger Murphy Biological Production Facility Ludwig Institute for Cancer Research Austin & Repatriation Medical Centre Studley Road, Heidelberg, Vic. 3084 Tel 61-3-94965463 Fax 61-3-94965436 Email murphy_r@licre.ludwig.edu.au From owner-proteins@net.bio.net Thu Oct 09 23:00:00 1997 Path: biosci!bloom-beacon.mit.edu!thetimes.pixel.kodak.com!news.kodak.com!news-pen-16.sprintlink.net!newsfeed.nysernet.net!news.nysernet.net!207.41.200.131!news-pen-1.sprintlink.net!news-east.sprintlink.net!news-dc-26.sprintlink.net!news-peer.sprintlink.net!news.sprintlink.net!Sprint!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news-feed.inet.tele.dk!news.misty.com!nntp.upenn.edu!taurus.fccc.edu!sauder From: sauder@polaris.fccc.edu (John Michael Sauder) Newsgroups: bionet.molbio.proteins Subject: Re: REfolding and the drama that is Date: 10 Oct 1997 22:21:42 GMT Organization: Fox Chase Cancer Center, Philadelphia, PA Lines: 35 Message-ID: <61m9pm$ugu$1@taurus.fccc.edu> References: <01bcd46d$38d8fe40$1f6f6682@default> NNTP-Posting-Host: polaris.fccc.edu In article <01bcd46d$38d8fe40$1f6f6682@default> "nH" writes: >Hello is there any info available on line >for refolding proteins in inclusion bodies in bacteria ? >I'm astudent..hence lack of knowledge and drive to look it up at the >library.. I did a quick search for "folding" and "inclusion bodies" on AltaVista and found 16,000 hits. Surely you are capable of doing a LITTLE bit of work? Well... maybe not. Since you obviously lack drive, here's a list of recent review articles from an OVID search on "folding" and "inclusion bod$". Guise AD. West SM. Chaudhuri JB. Protein folding in vivo and renaturation of recombinant proteins from inclusion bodies. [Review] Molecular Biotechnology. 6(1):53-64, 1996 Aug. Mukhopadhyay A. Inclusion bodies and purification of proteins in biologically active forms. [Review] Advances in Biochemical Engineering-Biotechnology. 56:61-109, 1997. Georgiou G. Valax P. Expression of correctly folded proteins in Escherichia coli. [Review] Current Opinion in Biotechnology. 7(2):190-7, 1996 Apr. King J. Haase-Pettingell C. Robinson AS. Speed M. Mitraki A. Thermolabile folding intermediates: inclusion body precursors and chaperonin substrates. [Review] FASEB Journal. 10(1):57-66, 1996 Jan. Rudolph R. Lilie H. In vitro folding of inclusion body proteins. [Review] FASEB Journal. 10(1):49-56, 1996 Jan. From owner-proteins@net.bio.net Thu Oct 09 23:00:00 1997 Path: biosci!GENOME.BIOTECH.WASHINGTON.EDU!eugene From: eugene@GENOME.BIOTECH.WASHINGTON.EDU (Eugene Kolker) Newsgroups: bionet.molbio.proteins Subject: Recomb98 deadline: Oct 20 Date: 10 Oct 1997 17:30:08 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 51 Sender: daemon@net.bio.net Distribution: world Message-ID: <199710110029.RAA21352@genome.biotech.washington.edu> NNTP-Posting-Host: net.bio.net CALL FOR PAPERS SECOND ANNUAL INTERNATIONAL CONFERENCE ON COMPUTATIONAL MOLECULAR BIOLOGY (RECOMB 98) March 22 - 25, 1998 New York City Sponsored by Association for Computing Machinery SIGACT with support from SLOAN Foundation US Department of Energy http://www.mssm.edu/biomath/recomb98.html The Second Annual Conference on Research in Computational Molecular Biology (RECOMB 98) will be held in New York City, March 22 - 25, 1998. Papers reporting on original research (both theoretical and experimental) in all areas of computational molecular biology are sought, including surveys of important recent results/directions. Typical but not exclusive topics of interest include: - Genomics - Molecular sequence analysis - Recognition of genes and regulatory elements - Molecular evolution - Protein structure - Combinatorial libraries and drug design ABSTRACT SUBMISSION: Authors are requested to send 10 copies (preferably two sided copies) of a detailed extended abstract (5-10 pages) to: Professor Pavel Pevzner RECOMB 98 Program Chair University of Southern California Department of Mathematics, DRB 155 Los Angeles, CA 90089-1113 An abstract must be received by October 20 (!), 1997. This is a firm deadline. Simultaneous submission to another conference or journal is allowed. For more information please visit the conference web page: http://www.mssm.edu/biomath/recomb98.html From owner-proteins@net.bio.net Thu Oct 09 23:00:00 1997 Path: biosci!daresbury!uninett.no!news.maxwell.syr.edu!newsxfer3.itd.umich.edu!oleane!jussieu.fr!univ-lille1.fr!cict.fr!ensat.fr!gerber From: gerber@ensat.fr Newsgroups: bionet.molbio.proteins Subject: micro two dimensional electrophoresis Date: Fri, 10 Oct 1997 09:23:38 UNDEFINED Lines: 47 Message-ID: NNTP-Posting-Host: 193.49.128.87 X-Newsreader: Trumpet for Windows [Version 1.0 Rev B final beta #1] I'm trying to analyse the total proteins contained in soybean seeds by micro two-dimensional electrophoresis, using Pharmacia Phastsystem. I have trouble to achieve it. Here are my recipies. Extraction : Urea 8M Pharmalyte 3-10, 2% DTT 5% Detergent 4% IEF : Acrylamide gel rehydrated with : Urea 8M Detergent (NP-40) 0.5% Pharmalyte 7% Equilibration : 2 minutes in 0.112M Tris 1% DTT 2.5% SDS and 2 minutes in 0.112M Tris 2.5% SDS 0.001% BPB Then SDS-PAGE Then coomassie or silver staining. The IEF seems to be correct, but the SDS-PAGE doesn't give proper results. With coomassie staining there is a strong blue wave on the migration front, the basic side doesn't seem to migrate correctly (no spot and the blue wave is bigger). I can only see spots on the middle of the gel, which correspond to storage protein. No spot on the first half of the gel. Can someone be of any help ? Thanks a lot Sophie Gerber gerber@ensat.fr From owner-proteins@net.bio.net Thu Oct 09 23:00:00 1997 Path: biosci!daresbury!uninett.no!newsfeed.nacamar.de!newscore.univie.ac.at!news-ge.switch.ch!news-zh.switch.ch!rzunews.unizh.ch!NewsWatcher!user From: zjons@vetbio.unizh.ch (Zophonias O. Jonsson) Newsgroups: bionet.molbio.proteins Subject: Re: Native protein purification Date: Fri, 10 Oct 1997 21:35:36 +0200 Organization: Universitat Zurich-Irchel Lines: 22 Message-ID: References: <343D20BE.669E@uni-konstanz.de> <343E64F0.497@nospam.ic.ac.uk> NNTP-Posting-Host: 130.60.120.27 In article <343E64F0.497@nospam.ic.ac.uk>, k.desmet@nospam.ic.ac.uk wrote: > Robert Deicher wrote: > > > Tris buffers interfere with binding, use phosphate instead. If you don't > want phosphate in your purified protein, bind it with phosphate, and > wash and elute in Tris (I haven't got around to trying other buffers > yet, but maybe somebody else in the Newsgroup has any tips forme here?). I regularly use HEPES for a second wash + elution. It works very well with both NTA and EDA. Zophonias _____________________________________________________________________ Zophonias O. Jonsson Institut fur Veterinarbiochemie Tel: (41-1)-635-54-75 Universitat Zurich-Irchel Fax: (41-1)-635-68-16 Winterthurerstrasse 190 CH-8057 Zurich Switzerland _____________________________________________________________________ From owner-proteins@net.bio.net Thu Oct 09 23:00:00 1997 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news-peer.gsl.net!news-stkh.gip.net!news.gsl.net!gip.net!news.kolumbus.fi!news.uninet.ee!kadri.ut.ee!tamm!tpungas From: tpungas@tamm.eenet.ee (Tanel Pungas) Newsgroups: bionet.molbio.proteins Subject: His-Tag and protein-protein interaction assay Date: 10 Oct 1997 19:02:10 GMT Organization: Estonian Biocentre Lines: 17 Message-ID: <61lu3i$5om$1@kadri.ut.ee> NNTP-Posting-Host: tamm.ebc.ee Mime-Version: 1.0 Content-Type: text/plain; charset=ISO-8859-1 Content-Transfer-Encoding: 8bit X-Trace: kadri.ut.ee 876510130 5910 (None) 193.40.23.2 X-Complaints-To: usenet@kadri.ut.ee X-Newsreader: TIN [version 1.2 PL2] Hello! Has anybody done in vitro protein-protein interaction assay with His-Tag proteins and in vitro translated proteins?Are there anykind of protocols or references about the binding contitions? With best regards, -- TANEL PUNGA, B.Sc. 23 Riia Street Fax:372-7-420286 Institute of Molecular Tel:372-7-420218 and Cell Biology E-mail:tpungas@ebc.ee EE2400 Tartu, ESTONIA From owner-proteins@net.bio.net Thu Oct 09 23:00:00 1997 Path: biosci!MAIL.CRYST.BBK.AC.UK!www From: www@MAIL.CRYST.BBK.AC.UK (Dave Houldershaw) Newsgroups: bionet.molbio.proteins Subject: Protein Crystallography on the Web Date: 10 Oct 1997 11:55:36 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 32 Sender: daemon@net.bio.net Distribution: world Message-ID: <343E7B22.5DAA4C68@mail.cryst.bbk.ac.uk> NNTP-Posting-Host: net.bio.net NEW Advanced Certificate in Protein Crystallography on the Web '97/'98 -------------------------------------------------------------------- Department of Crystallography, Birkbeck College, University of London Web site: http://px.cryst.bbk.ac.uk/ We are pleased to announce that we are now accepting registrations for this course. Protein crystallography is now a very multidisciplinary technique which has overlap with biochemistry, molecular biology, bioinformatics, biophysics and organic chemistry. The web course is therefore ideally suited for students of these disciplines who want to know in more detail (mainly non-mathematical) how structures are determined, how the quality of coordinates should be judged, and how crystallographic papers should be assessed. It will also appeal to those who envisage a further career in this exciting and rapidly expanding field, especially also in the pharmaceutical industry. This new course is delivered over the internet and makes use of innovative technologies including use of BioMOO for on-line tutorials, email discussion lists, and interactive course material. For more information please browse the URL given above. Please send any queries / requests for application forms to Claire Burton (c.burton@mail.cryst.bbk.ac.uk). The fee for students within the European Union is 260 pounds sterling, for non-EEA students the cost is 575 pounds sterling. Fee payments for overseas students should be made by cheque in pounds sterling drawn on a British bank. From owner-proteins@net.bio.net Thu Oct 09 23:00:00 1997 Path: biosci!daresbury!uninett.no!news.maxwell.syr.edu!news-peer.sprintlink.net!news-pull.sprintlink.net!news-in-east.sprintlink.net!news.sprintlink.net!Sprint!130.207.244.18!gatech!nntp-xfer.ncsu.edu!not-for-mail From: Brett Phinney Newsgroups: bionet.molbio.proteins Subject: Glass Wool Protein seperation Date: Fri, 10 Oct 1997 14:03:07 -0400 Organization: North Carolina State University Lines: 16 Message-ID: <343E6DDB.BB36EDB8@bchserver.bch.ncsu.edu> NNTP-Posting-Host: bch112.bch.ncsu.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.01 [en] (Win95; I) X-Priority: 3 (Normal) I'm trying to purify and separate two similar membrane proteins from each other. Back in the late 70's people had some success by running these proteins through a glass wool column. One protein apparently bound more tightly to the glass wool and was thus able to be separated from the other protein. Does anyone know what the mechanism for this is? How does the glass wool do this exactly? And can someone suggest a way to modernize this separation using HPLC. Thanks a lot Brett Phinney Biochemistry North Carolina State University From owner-proteins@net.bio.net Thu Oct 09 23:00:00 1997 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!rill.news.pipex.net!pipex!warm.news.pipex.net!pipex!join.news.pipex.net!pipex!server1.netnews.ja.net!server5.netnews.ja.net!server6.netnews.ja.net!server4.netnews.ja.net!news.cc.ic.ac.uk!not-for-mail From: Koen De Smet Newsgroups: bionet.molbio.proteins Subject: Re: Native protein purification Date: Fri, 10 Oct 1997 18:25:04 +0100 Organization: Department of Medical Microbiology Lines: 32 Message-ID: <343E64F0.497@nospam.ic.ac.uk> References: <343D20BE.669E@uni-konstanz.de> Reply-To: k.desmet@nospam.ic.ac.uk NNTP-Posting-Host: cb-22.sm.med.ic.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.02 (Macintosh; I; PPC) CC: Robert.Deicher@uni-konstanz.de Robert Deicher wrote: > > I am trying to purify a 6xHis tagged multi zinc finger protein from > baculovirus infected Sf9 cells under native conditions via Ni-NTA, > however the protein yield is extremely low. Any suggestions for pushing > up the protein yield? Should I first purify under denaturing conditions > and than try to refold? > Thanks for any help, Robert.Deicher@uni-konstanz.de Why is the yield low? Is it the expression level or loss during the purification? I purify 6xHis tagged proteins expressed at low level from bacteria, and found that the following are important: Forget about denaturing if you want a soluble, active protein. I tried this with six different proteins. Five of them precipitated and did not refold when I dialysed the urea away. The one that remained soluble had very little enymatic activity. The Ni++ binds His best at pH is 8.0 or higher. Tris buffers interfere with binding, use phosphate instead. If you don't want phosphate in your purified protein, bind it with phosphate, and wash and elute in Tris (I haven't got around to trying other buffers yet, but maybe somebody else in the Newsgroup has any tips forme here?). Always have at least 300 mM NaCl in your buffer. Koen De Smet ============================================================== ==>> To reply by email, remove "nospam." from the address <<== Imperial College School of Medicine at St Mary's http://www.sm.ic.ac.uk/medmicro/home. ============================================================== From owner-proteins@net.bio.net Thu Oct 09 23:00:00 1997 Path: biosci!daresbury!lyra.csx.cam.ac.uk!hgmp.mrc.ac.uk!not-for-mail From: p-jenner@nimr.mrc.ac.uk (Peter Jenner) Newsgroups: bionet.molbio.proteins Subject: Cytochrome oxidase Date: 10 Oct 1997 16:43:17 GMT Organization: NIMR Lines: 12 Message-ID: <61llv5$164$3@niobium.hgmp.mrc.ac.uk> NNTP-Posting-Host: nimsn52-0018.nimr.mrc.ac.uk X-Newsreader: WinVN 0.92.2 Does anyone know of a good source of cytochrome oxidase (bovine will do), other than Sigma or ICN. Both these suppliers' protein appear to be contaminated with BSA. We would like to generate some antibodies to as many of the subunits as possible, but need pure protein to get good antibodies. Thanks for any information Dr Peter Jenner NIMR London From owner-proteins@net.bio.net Thu Oct 09 23:00:00 1997 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!news-xfer.netaxs.com!news.his.com!news3.his.com!mail.his.com!dbunk From: "David M. Bunk" Newsgroups: bionet.molbio.proteins Subject: recombinant cardiac troponin I Date: Fri, 10 Oct 1997 09:08:52 -0400 Organization: Heller Information Services, Inc. Lines: 5 Message-ID: NNTP-Posting-Host: mail.his.com Mime-Version: 1.0 Content-Type: TEXT/PLAIN; charset=US-ASCII Does anyone know of a commercial source of human cardiac troponin I? All the commercial sources that I have found for this protein extract it from human hearts. The extracted protein is very expensive and in short supply. From owner-proteins@net.bio.net Thu Oct 09 23:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!europa.clark.net!4.1.16.34!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!Cabal.CESspool!bofh.vszbr.cz!lyra.csx.cam.ac.uk!server1.netnews.ja.net!warwick!leicester!usenet From: "Dr E. Buxbaum" Newsgroups: bionet.molbio.proteins Subject: Re: Efficient Protein Transfer Date: Fri, 10 Oct 1997 14:35:41 -0700 Organization: University of Leicester (PCFS User) Lines: 21 Message-ID: <343E9FAD.75C@le.ac.uk> References: <343DA667.ECE8013E@emory.edu> NNTP-Posting-Host: pc5.msb.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Win16; I) Rick Bright wrote: > > I am having trouble transferring enough myosin out of my 7.5% SDS-PAGE, > onto a Nylon membrane. I am using Tris-Glycine/10% MeOH transfer > buffer, equilibrating gel in same, activating membrane in 100% MeOH. I > have tried 3 hr transfer 250mA and overnight transfer 150mA. I am > staining the gel with Coumassie after transfer and have very large > myosin bands and no other bands. First I would try to leave out the methanol. Methanol is actually used to prevent small proteins from slipping through the membrane. With larger proteins it prevents their mobilisation from the gel. If that alone doesn't work, try addition of 0.1 mM SDS to your blotting buffer, which aids the mobilisation of large proteins but prevents binding of small proteins to the membrane. Methanol and SDS are therefore complementary. Personally, I have made very good experience with Dunn buffer (10 mM NaHCO3, 3 mM Na2CO3) instead of Towbin buffer. Gives slightly better results and is a lot cheaper. From owner-proteins@net.bio.net Thu Oct 09 23:00:00 1997 Path: biosci!fcs280s.ncifcrf.gov!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!cpk-news-hub1.bbnplanet.com!news.bbnplanet.com!Cabal.CESspool!bofh.vszbr.cz!lyra.csx.cam.ac.uk!server1.netnews.ja.net!warwick!leicester!usenet From: "Dr E. Buxbaum" Newsgroups: bionet.molbio.proteins Subject: Re: Native protein purification Date: Fri, 10 Oct 1997 14:28:45 -0700 Organization: University of Leicester (PCFS User) Lines: 16 Message-ID: <343E9E0D.784C@le.ac.uk> References: <343D20BE.669E@uni-konstanz.de> NNTP-Posting-Host: pc5.msb.le.ac.uk Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 3.01 (Win16; I) Robert Deicher wrote: > > I am trying to purify a 6xHis tagged multi zinc finger protein from > baculovirus infected Sf9 cells under native conditions via Ni-NTA, > however the protein yield is extremely low. Any suggestions for pushing > up the protein yield? This question can be answered with a clear: "That depends". First you have to find out why your yield is so low. Does your protein bind to the column (boil the beads in SDS sample buffer and see what you get in PAGE). If you get binding, which method did you use to elute your protein? The common imidazole gradient does not always work, try stripping the metal of the column with EDTA. If that doesn't work, your protein may denature on the column. Addition of urea or detergents may or may not help in this case, but it realy becomes a matter of trial and error. From owner-proteins@net.bio.net Thu Oct 09 23:00:00 1997 Path: biosci!BHAM.AC.UK!d.a.osullivan From: d.a.osullivan@BHAM.AC.UK ("Deirdre O'Sullivan") Newsgroups: bionet.molbio.proteins Subject: Peptide modelling Date: 10 Oct 1997 02:20:03 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 10 Sender: daemon@net.bio.net Distribution: world Message-ID: <1.5.4.16.19971010164143.304f9af2@sun1.bham.ac.uk> NNTP-Posting-Host: net.bio.net Hi, Does anyone know if there is any software out there to model structures of small peptides (<20 residues), and to calculate inter-residue distances ? TIA, Deirdre From owner-proteins@net.bio.net Thu Oct 09 23:00:00 1997 Path: biosci!OMRB.PNPI.SPB.RU!naryzhny From: naryzhny@OMRB.PNPI.SPB.RU ("Stanislav Naryzhny") Newsgroups: bionet.molbio.proteins Subject: sybscribe Date: 10 Oct 1997 01:41:52 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 1 Sender: daemon@net.bio.net Distribution: world Message-ID: <199710100841.LAA12058@omrb.pnpi.spb.ru> Reply-To: naryzhny@iname.com NNTP-Posting-Host: net.bio.net please, subscribe me From owner-proteins@net.bio.net Thu Oct 09 23:00:00 1997 Path: biosci!rutgers!gatech!192.26.210.166.MISMATCH!sunqbc.risq.qc.ca!newsxfer3.itd.umich.edu!news.mtu.edu!not-for-mail From: "Robert S. Donofrio" Newsgroups: bionet.molbio.proteins Subject: hydrocarbon transport Date: Fri, 10 Oct 1997 20:40:14 -0400 Organization: Michigan Technological University Lines: 11 Message-ID: <343ECAE8.781214A8@mtu.edu> NNTP-Posting-Host: bioserver.bio.mtu.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii; x-mac-type="54455854"; x-mac-creator="4D4F5353" Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 4.01 (Macintosh; I; PPC) X-Priority: 3 (Normal) I've just started researching the topic of bacterial transport for one of my classes. I am having a particularly difficult time finding info pertaining to the type(s) of transport system used by prokaryotes to translocate hydrocarbons, such as toluene, across the cell membrane. I was wondering if anyone could steer me in the right direction (i.e. proposed means of transport, published articles, etc.)? 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Osceola Pkwy Suite 242 Kissimmee Fl.34743 From owner-proteins@net.bio.net Thu Oct 09 23:00:00 1997 Path: biosci!rutgers!gatech!4.1.16.34.MISMATCH!cpk-news-hub1.bbnplanet.com!cpk-news-feed4.bbnplanet.com!cpk-news-feed1.bbnplanet.com!news.bbnplanet.com!nih.gov!not-for-mail From: jhmiller@helix.nih.gov (Jay Miller) Newsgroups: bionet.molbio.proteins Subject: Re: Cytochrome oxidase Date: 10 Oct 1997 19:56:37 GMT Organization: National Institutes of Health Lines: 18 Distribution: world Message-ID: <61m19l$pb1$1@light.nih.gov> References: <61llv5$164$3@niobium.hgmp.mrc.ac.uk> Reply-To: jhmiller@helix.nih.gov NNTP-Posting-Host: ams06057.niams.nih.gov X-Newsreader: IBM NewsReader/2 2.0 NIH had its annual instrument show this week. One of the booths featured a website called sciquest (www.sciquest.com). They are a search site with links to about 12,000 companies. The companies pay to be included. Try a search on it for cyt. ox. In <61llv5$164$3@niobium.hgmp.mrc.ac.uk>, p-jenner@nimr.mrc.ac.uk (Peter Jenner) writes: >Does anyone know of a good source of cytochrome oxidase (bovine will do), other than >Sigma or ICN. Both these suppliers' protein appear to be contaminated with BSA. We >would like to generate some antibodies to as many of the subunits as possible, but >need pure protein to get good antibodies. > >Thanks for any information > > >Dr Peter Jenner >NIMR >London > From owner-proteins@net.bio.net Thu Oct 09 23:00:00 1997 Path: biosci!bloom-beacon.mit.edu!howland.erols.net!news.maxwell.syr.edu!newsfeed.internetmci.com!131.103.1.114!iagnet.net!newsspool.doit.wisc.edu!news.itis.com!news.doit.wisc.edu!default From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: Native protein purification Date: Sat, 11 Oct 1997 03:45:46 GMT Organization: UW-Madison Lines: 15 Message-ID: <61mspa$tc_006@doit.wisc.edu> References: <343D20BE.669E@uni-konstanz.de> <343E64F0.497@nospam.ic.ac.uk> NNTP-Posting-Host: f182-124.net.wisc.edu X-No-Archive: Yes In article <343E64F0.497@nospam.ic.ac.uk>, k.desmet@nospam.ic.ac.uk wrote on Ni-NTA his-tag binding: #Tris buffers interfere with binding, use phosphate instead. If you don't #want phosphate in your purified protein, bind it with phosphate, and #wash and elute in Tris (I haven't got around to trying other buffers #yet, but maybe somebody else in the Newsgroup has any tips forme here?). #Always have at least 300 mM NaCl in your buffer. Cam anyone explain this for me? "Tris interfere". Why? How? Is this the case of poorly exposed tag or general? TIA, Dima From owner-proteins@net.bio.net Thu Oct 09 23:00:00 1997 Path: biosci!agate!howland.erols.net!iagnet.net!144.92.88.12!newsspool.doit.wisc.edu!news.itis.com!news.doit.wisc.edu!default From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: Glass Wool Protein seperation Date: Sat, 11 Oct 1997 03:36:56 GMT Organization: UW-Madison Lines: 24 Message-ID: <61ms8o$tc_002@doit.wisc.edu> References: <343E6DDB.BB36EDB8@bchserver.bch.ncsu.edu> NNTP-Posting-Host: f182-124.net.wisc.edu X-No-Archive: Yes In article <343E6DDB.BB36EDB8@bchserver.bch.ncsu.edu>, Brett Phinney wrote: #I'm trying to purify and separate two similar membrane proteins from #each other. Back in the late 70's people had some success by running #these proteins through a glass wool column. One protein apparently bound #more tightly to the glass wool and was thus able to be separated from #the other protein. Does anyone know what the mechanism for this is? How #does the glass wool do this exactly? And can someone suggest a way to #modernize this separation using HPLC. AFAIK glass-based separation is is a complex mix of charge/hydrophobic interactions. It is generally categorized as "absorption" chromatography which is a way of saying "unknown but works" (compare with "pseudo-affinity" used to described dye-based chromatography). Bottom line if you don't have the exactly the same wool, you stand almost no chance of success with any other. Separating just about any two proteins from one another should not be a problem. I am 100% positive it can be done with variety of techniques. What the proteins are like and how are they different? Dima From owner-proteins@net.bio.net Thu Oct 09 23:00:00 1997 Path: biosci!bloom-beacon.mit.edu!howland.erols.net!ais.net!iagnet.net!newsspool.doit.wisc.edu!news.itis.com!news.doit.wisc.edu!default From: klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) Newsgroups: bionet.molbio.proteins Subject: Re: His-Tag and protein-protein interaction assay Date: Sat, 11 Oct 1997 03:41:04 GMT Organization: UW-Madison Lines: 14 Message-ID: <61msgg$tc_004@doit.wisc.edu> References: <61lu3i$5om$1@kadri.ut.ee> NNTP-Posting-Host: f182-124.net.wisc.edu X-No-Archive: Yes In article <61lu3i$5om$1@kadri.ut.ee>, tpungas@tamm.eenet.ee (Tanel Pungas) wrote: # # Hello! # # Has anybody done in vitro protein-protein interaction assay with His-Tag #proteins and in vitro translated proteins?Are there anykind of protocols or #references about the binding contitions? As long as maintaning ~ 300 mM of salt is not a problem (far from physiological conditions and so the relevance of results has to be confirmed) AND 6xHis is fully exposed, the protocol should be no different from anything similar (GST fusions, etc). Dima From owner-proteins@net.bio.net Fri Oct 10 23:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!europa.clark.net!206.229.87.25!news-peer.sprintlink.net!news.sprintlink.net!Sprint!newsfeed.internetmci.com!198.60.22.3!xmission!news.cc.utah.edu!not-for-mail From: pjf Newsgroups: bionet.molbio.proteins Subject: Re: Native protein purification Date: 11 Oct 1997 00:21:23 -0600 Organization: planet of the monkey boys Lines: 24 Sender: zinc@zifi.genetics.utah.edu Message-ID: References: <343D20BE.669E@uni-konstanz.de> NNTP-Posting-Host: zifi.genetics.utah.edu To: Robert.Deicher@uni-konstanz.de X-Newsreader: Quassia Gnus v0.12/XEmacs 20.2 X-Face: &dh1aHu*v\s24A9~2qU[ln~;(Cz#X]d2r-@X>#j9U@vx!v8%IYU''n-H1D)}U(-0Te~ LD|Cyg6Gh-aFN{B*VSKhs}en{nXo3xkveE#BxR9 I am trying to purify a 6xHis tagged multi zinc finger protein from > baculovirus infected Sf9 cells under native conditions via Ni-NTA, > however the protein yield is extremely low. Any suggestions for pushing > up the protein yield? Should I first purify under denaturing conditions > and than try to refold? 1) yes. 2) grow more cells. do a damned 10 L prep if you have to. who cares? it's affinity chromatography so you can pump many liters of extract through the column and capture nearly all of the protein. rock on, -pjf, -- "There is only one aim in life and that is to live it." Karl Shapiro,(1959) from an essay on Henry Miller's Tropic of Cancer finger zinc-pgp@zifi.genetics.utah.edu for PGP key zifi runs Linux 2.1.56 http://zifi.genetics.utah.edu From owner-proteins@net.bio.net Fri Oct 10 23:00:00 1997 Path: biosci!bcm.tmc.edu!news.msfc.nasa.gov!newsfeed.internetmci.com!198.60.22.3!xmission!news.cc.utah.edu!not-for-mail From: pjf Newsgroups: bionet.molbio.proteins Subject: Re: Native protein purification Date: 11 Oct 1997 00:21:04 -0600 Organization: planet of the monkey boys Lines: 24 Sender: zinc@zifi.genetics.utah.edu Message-ID: References: <343D20BE.669E@uni-konstanz.de> NNTP-Posting-Host: zifi.genetics.utah.edu Mime-Version: 1.0 (generated by tm-edit 7.106) Content-Type: text/plain; charset=US-ASCII X-Newsreader: Quassia Gnus v0.12/XEmacs 20.2 X-Face: &dh1aHu*v\s24A9~2qU[ln~;(Cz#X]d2r-@X>#j9U@vx!v8%IYU''n-H1D)}U(-0Te~ LD|Cyg6Gh-aFN{B*VSKhs}en{nXo3xkveE#BxR9 I am trying to purify a 6xHis tagged multi zinc finger protein from > baculovirus infected Sf9 cells under native conditions via Ni-NTA, > however the protein yield is extremely low. Any suggestions for pushing > up the protein yield? Should I first purify under denaturing conditions > and than try to refold? 1) yes. 2) grow more cells. do a damned 10 L prep if you have to. who cares? it's affinity chromatography so you can pump many liters of extract through the column and capture nearly all of the protein. rock on, -pjf, -- "There is only one aim in life and that is to live it." Karl Shapiro,(1959) from an essay on Henry Miller's Tropic of Cancer finger zinc-pgp@zifi.genetics.utah.edu for PGP key zifi runs Linux 2.1.56 http://zifi.genetics.utah.edu From owner-proteins@net.bio.net Fri Oct 10 23:00:00 1997 Path: biosci!agate!spool.mu.edu!uwm.edu!news-out.internetmci.com!newsfeed.internetmci.com!198.60.22.3!xmission!news.cc.utah.edu!not-for-mail From: pjf Newsgroups: bionet.molbio.proteins Subject: Re: Protein Assay Choice (need assistance) Date: 11 Oct 1997 00:25:09 -0600 Organization: planet of the monkey boys Lines: 6 Sender: zinc@zifi.genetics.utah.edu Message-ID: References: <61boiq$omk$1@piglet.cc.uic.edu> NNTP-Posting-Host: zifi.genetics.utah.edu X-Newsreader: Quassia Gnus v0.12/XEmacs 20.2 X-Face: &dh1aHu*v\s24A9~2qU[ln~;(Cz#X]d2r-@X>#j9U@vx!v8%IYU''n-H1D)}U(-0Te~ LD|Cyg6Gh-aFN{B*VSKhs}en{nXo3xkveE#BxR9 Newsgroups: bionet.molbio.proteins Subject: Re: Protein Purification Systems Date: 11 Oct 1997 10:35:24 -0600 Organization: planet of the monkey boys Lines: 22 Sender: zinc@zifi.genetics.utah.edu Message-ID: References: <199710091605.LAA27998@cnas.smsu.edu> NNTP-Posting-Host: zifi.genetics.utah.edu Mime-Version: 1.0 (generated by tm-edit 7.106) Content-Type: text/plain; charset=US-ASCII To: dac012f@CNAS.SMSU.EDU (Dean Cuebas) X-Newsreader: Quassia Gnus v0.12/XEmacs 20.2 X-Face: &dh1aHu*v\s24A9~2qU[ln~;(Cz#X]d2r-@X>#j9U@vx!v8%IYU''n-H1D)}U(-0Te~ LD|Cyg6Gh-aFN{B*VSKhs}en{nXo3xkveE#BxR9 I am in the market for a protein purification system and I had > demonstrations for the Perseptive Sprint system and the comparable AKTA > system, and felt that the AKTA software was far superior, but the > replication of results (actual performance) on the Perseptive system was > far superior. -- "There is only one aim in life and that is to live it." Karl Shapiro,(1959) from an essay on Henry Miller's Tropic of Cancer finger zinc-pgp@zifi.genetics.utah.edu for PGP key zifi runs Linux 2.1.56 http://zifi.genetics.utah.edu From owner-proteins@net.bio.net Sat Oct 11 23:00:00 1997 Path: biosci!daresbury!uninett.no!news.algonet.se!newsfeed.direct.ca!newshub1.home.com!news.home.com!xfer.kren.nm.kr!ccsun2.sogang.ac.kr!not-for-mail From: "YongWan Kim" Newsgroups: bionet.molbio.proteins Subject: sds-page Date: 12 Oct 1997 09:03:11 GMT Organization: crystal_lab Lines: 18 Message-ID: <01bcd6ee$df077660$8623efa3@cryst4.sogang.ac.kr> NNTP-Posting-Host: cryst4.sogang.ac.kr X-Newsreader: Microsoft Internet News 4.70.1161 Hi, all I am a student. I have purified the intracrystalline macromolecules from the shell of invertebrate. By gel-filtration I know roughly these contain at least more than three different classes of macromolecules, 160kD, 50kD, 20kD. So I decided to use the sds-page. In the manuals of Bio-Rad, I treid to find any peak, but no gain. So I tried 7%, 12%, 15%, and 20% gel concentration. From all conditions mentioned above, I found that at the bottom of gel, some band was located. That is, everything was not eluted. Frankly I really don't understand. Why not? Thanks, Kim From owner-proteins@net.bio.net Sun Oct 12 23:00:00 1997 From: krasel@wpxx02.toxi.uni-wuerzburg.de (Cornelius Krasel) Newsgroups: bionet.molbio.proteins Subject: Re: Native protein purification Date: Sun, 12 Oct 1997 15:09:25 +0200 Organization: University of Wuerzburg, Germany Lines: 24 Message-ID: <56iq16.m5.ln@wpxx02.toxi.uni-wuerzburg.de> References: <343D20BE.669E@uni-konstanz.de> <343E64F0.497@nospam.ic.ac.uk> <61mspa$tc_006@doit.wisc.edu> NNTP-Posting-Host: wpxx02.toxi.uni-wuerzburg.de X-Newsreader: TIN [UNIX 1.3 950824BETA PL0] Path: biosci!news.ic.sunysb.edu!news-pen-15.sprintlink.net!newsfeed.nysernet.net!news.nysernet.net!204.92.55.84!feed.nntp.acc.ca!128.230.129.112.MISMATCH!news-spur1.maxwell.syr.edu!news.maxwell.syr.edu!news-peer.sprintlink.net!news.sprintlink.net!Sprint!newsfeed.internetmci.com!194.162.162.196!newsfeed.nacamar.de!uni-erlangen.de!winx03!wpxx02!not-for-mail Dima Klenchin (klenchin@facstaff.REMOVE_TO_REPLY.wisc.edu) wrote: > In article <343E64F0.497@nospam.ic.ac.uk>, k.desmet@nospam.ic.ac.uk wrote > on Ni-NTA his-tag binding: > #Tris buffers interfere with binding, use phosphate instead. If you don't > #want phosphate in your purified protein, bind it with phosphate, and > #wash and elute in Tris (I haven't got around to trying other buffers > #yet, but maybe somebody else in the Newsgroup has any tips forme here?). > #Always have at least 300 mM NaCl in your buffer. > > Cam anyone explain this for me? "Tris interfere". Why? How? I believe (from own experience) that Tris leaches the Nickel from the resin. Same goes for other Good buffers. (It has nothing to do with the His-tag.) Short exposures to low Tris concentrations do not matter very much. (Quiagen recommends Tris <= 20 mM, if I recall correctly.) --Cornelius. -- /* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */ /* D-97078 Wuerzburg, Germany email: phak004@rzbox.uni-wuerzburg.de SP4 */ /* "Science is the game we play with God to find out what His rules are." */ From owner-proteins@