From Victor_Morales from URMC.Rochester.edu Sat Feb 2 16:30:54 2008 From: Victor_Morales from URMC.Rochester.edu (Morales, Victor) Date: Sat Feb 2 20:01:38 2008 Subject: [Protein-analysis] Protocol for human CD38 WB In-Reply-To: <200802021705.m12H5NL25154@net.bio.net> References: <200802021705.m12H5NL25154@net.bio.net> Message-ID: <8EC360C02780954D90DFEBB9B622ADBF049483@MEDMAIL.urmc-sh.rochester.edu> Connie: Have your mab's been tested for western blotting? Some antibodies only recognize the native protein conformation and cannot bind to the denatured forms in the western blot. Victor -----Original Message----- From: proteins-bounces@oat.bio.indiana.edu [mailto:proteins-bounces@oat.bio.indiana.edu] On Behalf Of proteins-request@oat.bio.indiana.edu Sent: Saturday, February 02, 2008 12:05 PM To: proteins@magpie.bio.indiana.edu Subject: Proteins Digest, Vol 33, Issue 1 Send Proteins mailing list submissions to proteins@net.bio.net To subscribe or unsubscribe via the World Wide Web, visit http://www.bio.net/biomail/listinfo/proteins or, via email, send a message with subject or body 'help' to proteins-request@net.bio.net You can reach the person managing the list at proteins-owner@net.bio.net When replying, please edit your Subject line so it is more specific than "Re: Contents of Proteins digest..." Today's Topics: 1. Requesting protocol for human CD38 western analysis (Connie Lam) ---------------------------------------------------------------------- Message: 1 Date: Fri, 1 Feb 2008 12:43:19 +0800 From: "Connie Lam" Subject: [Protein-analysis] Requesting protocol for human CD38 western analysis To: proteins@magpie.bio.indiana.edu Message-ID: <93f327860801312043q76210882xba9eb6ad9c5b0512@mail.gmail.com> Content-Type: text/plain; charset=ISO-8859-1 Dear all, *Requesting protocol for human CD38 western analysis* I are studying human CD38 with different hCD38 monoclonal antibody T16/IB4. I can use the Ab to stain hCD38-transfected HEK293 cells. When I used T16/IB4 for western, no bands were observed. Here is my protocol. I am using the RIPA buffer + protease inhibitor to lyse the cells and run on 10% SDS-PAGE gel. After transfered onto nitrocellulose membrane and blocked with 5% skim milk, the membrane was incubated with one of the Ab (4ug/ml) overnight at 4oC followed by anti-mouse-HRP for 1 h at room temperature. I am looking for other protocols for sample preparation and western for hCD38? Thanks. Connie ------------------------------ _______________________________________________ Proteins mailing list Proteins@net.bio.net http://www.bio.net/biomail/listinfo/proteins End of Proteins Digest, Vol 33, Issue 1 *************************************** From ladyhawke72 from gmail.com Mon Feb 4 03:48:23 2008 From: ladyhawke72 from gmail.com (marie logan) Date: Mon Feb 4 11:21:47 2008 Subject: [Protein-analysis] BUFFALO MILK PROTEINS Message-ID: <4d1277fa0802040048o5bb44558nd8967b278b514828@mail.gmail.com> Hi just a couple of lines to ask some help. can anyone please tell me where to find HPLC on buffalo milk proteins (quantification, identification of caseins etc)? Can anyone please help me??Are there any articles ? Am despereate, dont know where 2 look. Thanks for you help From nicolas.bazeille from curie.u-psud.fr Mon Feb 4 10:39:41 2008 From: nicolas.bazeille from curie.u-psud.fr (Nicolas BAZEILLE) Date: Mon Feb 4 11:21:58 2008 Subject: [Protein-analysis] Is there a way to enhance yield of soluble DNA-binding protein in limiting DNA-protein interactions ? Message-ID: <6.1.1.1.2.20080204140334.01b11990@pop.curie.u-psud.fr> hi, I try to purify a protein from yeast to assay some structural studies. unfortunatly, the yield is really poor and I want to find some ways to increase it. First, I change the medium and the temperature and the time of induction but I think that now it is optimal. Because my protein is a DNA binding protein, I think that I lost some proteins fixed on genomic DNA that are found in the pellet during the step of clarification just after breaking cells. Does it may be right ? Is there a way to prevent this interactions (high salt concentration, additives ??) I ask also, to know if there is a special manipulation to get a clear pellet during the step of cells collecting because my pellet is rapidly resolublize and the end of the run in spite of an optimal speed, resulting in the loss of some cells. Is there specials additives to get clear, compact and well packed pellet of cells, that I can put directly on the medium (YNB -URA galactose) at the end of induction. Nicolas pHD student Institut Curie Paris france From clement from bio.mls.eng.osaka-u.ac.jp Mon Feb 4 20:22:26 2008 From: clement from bio.mls.eng.osaka-u.ac.jp (Clement Angkawidjaja) Date: Mon Feb 4 20:36:59 2008 Subject: [Protein-analysis] Is there a way to enhance yield of soluble DNA-binding protein in limiting DNA-protein interactions ? References: <200802041703.m14H3WL09998@net.bio.net> Message-ID: <000b01c86795$915e17f0$3100a8c0@CLEMENT> Do SDS-PAGE(s) to see whether your protein is expressed well and to know where it is. It's that simple. In your protein sequence, did you include the signal sequence for import into the nucleus? If you did not, you should not expect import. What kind of lysis method you used? In many cases, DNA-protein interaction is stable under high salt. However, it depends on what kind of interaction your protein and DNA has. Yeast cells should stay in the pellet nicely. If yours is easily solubilized, perhaps your centrifuge speed is too low OR something happened by overexpressing your protein... (which should be interesting... I don't know. I'm not a yeast expert). Clement Angkawidjaja, Ph.D Laboratory of Molecular Biotechnology (Kanaya Laboratory) Division of Advanced Science and Biotechnology Graduate School of Engineering, Osaka University 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan Tel./ Fax: +81-6-6879-4580 > Date: Mon, 04 Feb 2008 16:39:41 +0100 > From: Nicolas BAZEILLE > Subject: [Protein-analysis] Is there a way to enhance yield of soluble > DNA-binding protein in limiting DNA-protein interactions ? > To: proteins@magpie.bio.indiana.edu > Message-ID: <6.1.1.1.2.20080204140334.01b11990@pop.curie.u-psud.fr> > Content-Type: text/plain; charset="us-ascii"; format=flowed > > hi, > > > I try to purify a protein from yeast to assay some structural studies. > unfortunatly, the yield is really poor and I want to find some ways to > increase it. First, I change the medium and the temperature and the time > of > induction but I think that now it is optimal. > Because my protein is a DNA binding protein, I think that I lost some > proteins fixed on genomic DNA that are found in the pellet during the step > of clarification just after breaking cells. Does it may be right ? Is > there > a way to prevent this interactions (high salt concentration, additives ??) > I ask also, to know if there is a special manipulation to get a clear > pellet during the step of cells collecting because my pellet is rapidly > resolublize and the end of the run in spite of an optimal speed, resulting > in the loss of some cells. Is there specials additives to get clear, > compact and well packed pellet of cells, that I can put directly on the > medium (YNB -URA galactose) at the end of induction. > > > Nicolas pHD student Institut Curie Paris france > > > > > ------------------------------ > > _______________________________________________ > Proteins mailing list > Proteins@net.bio.net > http://www.bio.net/biomail/listinfo/proteins > > End of Proteins Digest, Vol 33, Issue 3 > *************************************** From ksalmon from uci.edu Wed Feb 6 14:21:16 2008 From: ksalmon from uci.edu (Kirsty Salmon) Date: Wed Feb 6 19:25:46 2008 Subject: [Protein-analysis] xylose isomerase? Message-ID: <003901c868f5$72169d20$5643d760$@edu> Did you ever figure out where to purchase xylose isomerase? We are also looking. Thanks, Kirsty Kirsty A. Salmon, Ph.D. Manager/Project Scientist II Institute for Genomics & Bioinformatics University of California, Irvine Room 1309, CAL(IT)2 Campus Dr. Irvine, CA 92697-2800 ksalmon@uci.edu From sticher from bioc.unizh.ch Mon Feb 11 03:31:25 2008 From: sticher from bioc.unizh.ch (Patrick Sticher) Date: Mon Feb 11 13:49:34 2008 Subject: [Protein-analysis] First Announcement: 6th International NCCR Symposium on New Trends in Structural Biology, September 8 + 9, 2008 Message-ID: <47B007DD.6040101@bioc.unizh.ch> Dear colleagues, it is our pleasure to announce the 6th International NCCR Symposium taking place this September. First Announcement: 6th INTERNATIONAL NCCR SYMPOSIUM ON NEW TRENDS IN STRUCTURAL BIOLOGY September 8 + 9, 2008 Lecture Hall KOH B10, University of Zurich, Switzerland Confirmed speakers to date: Stephen Kowalczykowski ? Keiichi Namba ? Poul Nissen ? Andrej Sali ? A. Joshua Wand www.structuralbiology.uzh.ch/symposium2008.asp The registration slot opens end of March. Online registration will be possible directly from the above mentioned web site. The symposium will be followed directly by the Annual Meeting of the Swiss Society for Crystallography with two or three additional short lectures. Participants of the NCCR Symposium are invited to join this event as well. We do hope that this conference is of interest to you and would be pleased to welcome you in Zurich this fall. With best regards, Patrick Sticher _________________________________ Visit the NCCR on the Internet www.structuralbiology.uzh.ch Dr. Patrick Sticher Moser NCCR Scientific Officer Institute of Biochemistry University of Z?rich Winterthurerstrasse 190 CH - 8057 Z?rich Phone +41 / (0)44 / 635 54 84 Fax +41 / (0)44 / 635 59 08 Mail sticher@bioc.uzh.ch From d_lobby006 from yahoo.com Tue Feb 12 14:45:52 2008 From: d_lobby006 from yahoo.com (dobby) Date: Tue Feb 12 14:54:46 2008 Subject: [Protein-analysis] protein structure Message-ID: <9069a277-a489-487d-99f0-aeddf4b8326f@s19g2000prg.googlegroups.com> Good day i just want to knoe hoe to determine the primary,secondary and teriatry structure of haemoglobin or any tertiary structure of proyein.and the interaction and factors involved in the conformation at different level of conformation From brighttomorrow from gmail.com Thu Feb 14 19:21:21 2008 From: brighttomorrow from gmail.com (Nico) Date: Thu Feb 14 19:35:40 2008 Subject: [Protein-analysis] Recombinant Protein precipitate out under 20 mM KPB/100 mM NaCl ??? Message-ID: <200802141921212505281@gmail.com> Hi, I am changing buffer for my protein, which was purified from Ni-NTA column. Under 20mM KPB/500mM NaCl, it was good. But when change to 20 mM KPB/100 mM NaCl, the protein precipitate out. What should I do? Change to Tris-HCl Buffer or..... Please help me out, Many thanks, Nico From engelbert_buxbaum from hotmail.com Mon Feb 18 16:32:44 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Mon Feb 18 17:38:12 2008 Subject: [Protein-analysis] Re: protein structure References: <9069a277-a489-487d-99f0-aeddf4b8326f@s19g2000prg.googlegroups.com> Message-ID: Am 12.02.2008, 15:45 Uhr, schrieb dobby : > Good day > i just want to knoe hoe to determine the primary,secondary and > teriatry structure of haemoglobin or any tertiary structure of > proyein.and the interaction and factors involved in the conformation > at different level of conformation Any textbook on proteins, including (if I may say so) my own, includes a brief discussion about that subject. If you need more details a textbook on molecular biophysics would help, e.g. @book{Gla-00, AUTHOR= {R. Glaser}, TITLE= {Biophysics}, YEAR= {2000}, PUBLISHER= {Springer}, ADDRESS= {Berlin}, EDITION= {Fifth}, LANGUAGE= {engl} } From engelbert_buxbaum from hotmail.com Mon Feb 18 16:37:39 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Mon Feb 18 17:38:25 2008 Subject: [Protein-analysis] Re: Recombinant Protein precipitate out under 20 mM KPB/100 mM NaCl ??? References: Message-ID: Am 14.02.2008, 20:21 Uhr, schrieb Nico : > Under 20mM KPB/500mM NaCl, it was good. But when change to 20 mM KPB/100 > mM NaCl, > the protein precipitate out. What should I do? Change to Tris-HCl Buffer > or..... Looks like your protein is an albumin, with elongated shape and asymetric charge distribution. At low ionic strength they form head-to-tail aggregates, which precipitate. You can either keep the ionic strength high or change the pH and hence the charges on the protein. From incrediblyhigh from hotmail.com Wed Feb 20 09:14:55 2008 From: incrediblyhigh from hotmail.com (incrediblyhigh@hotmail.com) Date: Wed Feb 20 12:54:24 2008 Subject: [Protein-analysis] Re: Recombinant Protein precipitate out under 20 mM KPB/100 mM NaCl ??? References: Message-ID: <016263ae-23b6-43c5-901e-a651e7714cf6@e6g2000prf.googlegroups.com> On Feb 18, 9:37?pm, "Dr Engelbert Buxbaum" wrote: > Am 14.02.2008, 20:21 Uhr, schrieb Nico : > > > Under 20mM KPB/500mM NaCl, it was good. But when change to 20 mM KPB/100 ? > > mM NaCl, > > the protein precipitate out. What should I do? Change to Tris-HCl Buffer ? > > or..... > > Looks like your protein is an albumin, with elongated shape and asymetric ? > charge distribution. At low ionic strength they form head-to-tail ? > aggregates, which precipitate. You can either keep the ionic strength high ? > or change the pH and hence the charges on the protein. I agree with Dr Buxbaum. Try increasing the NaCl concentration (you could try between 200-500mM if you really need to drop the concentration), clearly the main reason for the precipitation. Changing the buffer/pH will probably not help much... From incrediblyhigh from hotmail.com Wed Feb 20 09:28:21 2008 From: incrediblyhigh from hotmail.com (incrediblyhigh@hotmail.com) Date: Wed Feb 20 12:54:32 2008 Subject: [Protein-analysis] Re: Is there a way to enhance yield of soluble DNA-binding protein in limiting DNA-protein interactions ? References: Message-ID: On Feb 4, 3:39?pm, Nicolas BAZEILLE wrote: > hi, > > I try to purify a protein from yeast to assay some structural studies. > unfortunatly, the yield is really poor and I want to find some ways to > increase it. First, I change the medium and the temperature and the time of > induction but I think that now it is optimal. > Because my protein is a DNA binding protein, I think that I lost some > proteins fixed on genomic DNA that are found in the pellet during the step > of clarification just after breaking cells. Does it may be right ? Is there > a way to prevent this interactions (high salt concentration, additives ??) > I ask also, to know if there is a special manipulation to get a clear > pellet during the step of cells collecting because my pellet is rapidly > resolublize and the end of the run in spite of an optimal speed, resulting > in the loss of some cells. Is there specials additives to get clear, > compact and well packed pellet of cells, that I can put directly on the > medium (YNB -URA galactose) at the end of induction. > > Nicolas pHD student Institut Curie Paris france Why not add some DNase to you lysis solution?? That should get rid of most DNA. Not having worked with yeast, can you sonicate the cells? That should also help in shearing the DNA. Can't help you with the cell pelleting problem I'm afraid... I agree with clement in that you should first look at total expression but maybe you already have: is the yield poor after purification or do you simply have low levels of expression? Assuming the problem lies with purification: High salt can help... in fact, the low yield may also be a consequence of not using the right buffer conditions for your protein. Ideally you would do some small scale lysis/purification experiments to determine the optimum pH and concentration of salt for your protein during lysis and subsequent steps. Does it have cysteines? If so, adding reducing agents might help... Low quantities of detergent (eg triton x100 or tween20, 0.01% or less) can help as well... From incrediblyhigh from hotmail.com Wed Feb 20 09:39:37 2008 From: incrediblyhigh from hotmail.com (incrediblyhigh@hotmail.com) Date: Wed Feb 20 12:54:37 2008 Subject: [Protein-analysis] Re: His-tagged protein purification from yeast (k lactis) supernatant References: <6ecae0f2-19ab-4360-8ec6-0b1fa43df351@x69g2000hsx.googlegroups.com> Message-ID: <7070a256-d654-404a-baae-7c0626207f8f@o77g2000hsf.googlegroups.com> On Jan 4, 1:08?am, Tiwari wrote: > The media contains no EDTA/EGTA/DDT/BME nothing just yeast extract + > peptones + galactose. Media alone was able to chelate Ni from resin. > > On Jan 3, 5:47 pm, d...@no.email.thankstospam.net (DK) wrote: > > > In article <6ecae0f2-19ab-4360-8ec6-0b1fa43df...@x69g2000hsx.googlegroups.com>, Tiwari wrote: > > > >Hi Guys, > > > >I am expressing the recombinant protein 6xHis tagged at C' terminal > > >secreting in YP-Galactose media by K lactis. My problem is whenever i > > >try to purify by using Ni-NTA column, it turns the column white as if > > >supernatant is chelating Ni from the column. I tried to condition > > >supernatant by adjusting pH 8.0 and Salt upto 1M with no success. > > >Please suggest me what should i do. Ammonium sulphate precipitation > > >result in inactivation of protein. i cant do dialysis economically to > > >huge culture volume. ?so these will not be a good option for > > >conditioning media. > > > Try adding 10 mM Mg2+. If you are lucky , the thing that strips Ni2+ > > also binds Mg2+. > > > Besides AS precipitation, another option to concentrate proteins > > and get rid of the media components that strip nickel is > > hydroxyapatite absorption. If "your" protein binds to an ion exchanger > > under medium's salt condition, that's also a good high capacity > > option (even if it does not, diluting medium with water before ion > > exchanger absorption is yet another option). > > > DK > > > >Thanks! Have you tried talon beads? They might perform better... What about changing the tag to say, a strep tag? From agarkarvi from yahoo.com Thu Feb 21 19:32:30 2008 From: agarkarvi from yahoo.com (Vinod Agarkar) Date: Fri Feb 22 09:05:37 2008 Subject: [Protein-analysis] Recombinant Protein precipitate out under 20 mM KPB/100 mM NaCl ??? Message-ID: <176944.50704.qm@web36609.mail.mud.yahoo.com> Hi, May be you could try followings: 1) While collecting fractions from Ni-NTA column, keep 0.25 to 0.5mM EDTA in collection tubes. 2) Dialyse against the buffer (elution buffer) for 2-3 hrs. 3) Now, make 20mM KPB and add slowly with intervals 20-30min to decrease the NaCl concentration (I will prefer to do in cold). You will know where your protein is happy, I mean what salt concentrations. 4) Changing buffer like Tris or anything is not a bad idea. 5) Check with KCl instead of NaCl or combinations of two.Or may be you need some other additives. Have a nice time. Vinod Agarkar Nico wrote: Hi, I am changing buffer for my protein, which was purified from Ni-NTA column. Under 20mM KPB/500mM NaCl, it was good. But when change to 20 mM KPB/100 mM NaCl, the protein precipitate out. What should I do? Change to Tris-HCl Buffer or..... Please help me out, Many thanks, Nico _______________________________________________ Proteins mailing list Proteins@net.bio.net http://www.bio.net/biomail/listinfo/proteins --------------------------------- Never miss a thing. Make Yahoo your homepage. --------------------------------- Never miss a thing. Make Yahoo your homepage. From a.r.mcewan from abdn.ac.uk Mon Feb 25 08:27:33 2008 From: a.r.mcewan from abdn.ac.uk (Mcewan, Dr Andrew) Date: Mon Feb 25 12:48:29 2008 Subject: [Protein-analysis] (no subject) Message-ID: proteins@magpie.bio.indiana.edu Slightly offtopic... Sorry but I'm doing portein binding assays (so close enough right?) Does anyone know an alternative to AIDA phosphorimager software (that I can use on any computer - without a dongle - crypt key) i.e is free to download. As I'm getting in the way of others trying to use the machine while I attempt to process the images!!! Thanksin advance, Andy M From engelbert_buxbaum from hotmail.com Wed Feb 27 13:46:55 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Wed Feb 27 22:12:26 2008 Subject: [Protein-analysis] Re: (no subject) References: Message-ID: Am 25.02.2008, 09:27 Uhr, schrieb Mcewan, Dr Andrew : > Does anyone know an alternative to AIDA phosphorimager software (that I > can use on any computer - without a dongle - crypt key) i.e is free to > download. > > As I'm getting in the way of others trying to use the machine while I > attempt to process the images!!! If you have a spot pattern, you could use NIH Image for evaluation. Originally a Mac program, but a Wintel version is available from http://www.scioncorp.com/