From kstiwari from gmail.com Thu Jan 3 00:44:07 2008 From: kstiwari from gmail.com (Tiwari) Date: Thu Jan 3 16:05:06 2008 Subject: [Protein-analysis] His-tagged protein purification from yeast (k lactis) supernatant Message-ID: <6ecae0f2-19ab-4360-8ec6-0b1fa43df351@x69g2000hsx.googlegroups.com> 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. Thanks! From melissa.mazan from tufts.edu Thu Jan 3 12:19:11 2008 From: melissa.mazan from tufts.edu (Melissa Mazan) Date: Thu Jan 3 16:05:16 2008 Subject: [Protein-analysis] Western blot using FACS cells Message-ID: <477D190F.1040705@tufts.edu> Hi all - I am trying to identify proteins using WB on cells that have already been through FACs. We make a single cell suspension from lung, bring it on ice to our core facility (about 45 minutes away) and then bring the sorted cells back to the lab in media on ice. About 4-5 hours in all. The sorted cells I receive range from 1200 cells/ 250 ul to 1x10 to the 7th cells. I wash the cells at slow speed in PBS (4C) when I receive them, then extract protein using M-Per from Pierce. I'm not having much luck with samples that have less than a million cells. Any suggestions? Many thanks - Melissa -- Melissa R. Mazan, DVM, Diplomate ACVIM Associate Professor and Director of Sports Medicine Tufts Cummings School of Veterinary Medicine 200 Westboro Road North Grafton, MA 01536 508-887-4589 From kstiwari from gmail.com Thu Jan 3 20:08:40 2008 From: kstiwari from gmail.com (Tiwari) Date: Thu Jan 3 20:38:47 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: 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! From sandy_zhu_2006 from hotmail.com Thu Jan 10 00:01:30 2008 From: sandy_zhu_2006 from hotmail.com (sandy_zhu_2006@hotmail.com) Date: Thu Jan 10 10:25:53 2008 Subject: [Protein-analysis] problem of concentrating protein Message-ID: The protein I am purifying is a kind of zinc finger protein which has 81 amino acid and its theoretical pI is 9.19.But after purified with Hitrap Heparin column the target protein were still stable,then when I use the binding buffer(20mM Tris,10mM Mer,100mM NaCl and 1mM ZnCl2,pH 7.0)to dialyze in order to remove the 2M NaCl which were used in eluting the protein from the Heparin column, I found that there are many sediment in it. How to solve this problem?Is there any buffer which is more siutable than Tris? Since at pH7.0 Tris is not as efficient as it was. Plus I tried phosphate buffer,but it will interact with the ion zinc (within zinc finger protein) and produce sendiment too. From asanchez from bio.puc.cl Thu Jan 10 15:07:12 2008 From: asanchez from bio.puc.cl (Alejandro Sanchez) Date: Thu Jan 10 15:30:39 2008 Subject: [Protein-analysis] Two bands on p53 WB. Message-ID: <6.1.2.0.2.20080110164951.01b42600@localhost> Hello I?m performing western blots on lysates from EBV transformed lymphocytes. I electrophorese the lysates on a 12% SDS PAGE and the incubation with the anti p53 DO1 shows a double band in some individuals and only one band in others, the difference between the bands is approx 3-4 kDa. This happens without any treatment. Some one has experienced this???. Is this due to phosphorylation???, p53 isoforms differentially expressed in different individuals???. I?ll appreciate any suggestion, Alejandro From bell.eapen from gmail.com Fri Jan 11 03:07:45 2008 From: bell.eapen from gmail.com (Bell) Date: Fri Jan 11 12:56:45 2008 Subject: [Protein-analysis] Protein-Protein Docking Problem. Message-ID: I am looking for a solution to the following problem. Any insight will be greatly appreciated. A membrane receptor (A) has an extracellular domain (AE), transmembrane domain (AT) and intracellular domain (AI). A bacteria (B) binds to (AE) leading to dimerization of (A) at (AT) and subsequent downstream signaling through (AI). (A) has no known natural ligands. (A) has one known inhibitor (I) binding to (AE) The structure of (AE) bound to (I) is available from PDB. How do we identify which protein in (B) binds to (AE). The obvious solution is to dock all proteins with known structures in (B) to all known pockets in AE. Any better solutions? Thanks Bell Eapen GulfDoctor.net Dermatologist.co.in From dogoyi from mail.uonbi.ac.ke Sat Jan 12 07:05:29 2008 From: dogoyi from mail.uonbi.ac.ke (dogoyi@mail.uonbi.ac.ke) Date: Sat Jan 12 12:59:55 2008 Subject: [Protein-analysis] 2-D analysis software Message-ID: <55025.10.2.21.43.1200139529.squirrel@mail.uonbi.ac.ke> Hallo, Anybody out there with an idear of software available over the internet that may help us do some analysis of differentially expressed proteins through 2-D? We have tried "Flicker" unfortunately it seems the format in which we have saved our photos is not compatible. We would really appreciate any suggestion. D.O. Ogoyi University of Nairobi, Dept. of Biochemistry P.O. BOX 30197, 00100 NAIROBI, KENYA From jepoirrier from gmail.com Sun Jan 13 13:20:04 2008 From: jepoirrier from gmail.com (Jean-Etienne Poirrier) Date: Sun Jan 13 20:01:32 2008 Subject: [Protein-analysis] Re: 2-D analysis software Message-ID: <7e4cff80801131020o21108c0fk6b04569434b4741c@mail.gmail.com> Hi, > Anybody out there with an idear of software available over the internet > that may help us do some analysis of differentially expressed proteins > through 2-D? We have tried "Flicker" unfortunately it seems the format in > which we have saved our photos is not compatible. We would really > appreciate any suggestion. I'm afraid there is no such software (at least for free). Melanie, Delta2D, Progenesis, ImageMaster, DeCyder, PDQuest, ... are all offline and commercial (and very expensive) software [1]. I only know 3 other software that were available on the web: - Gellab [2] but it doesn't seem to be maintained nor available now (afaik) - The Open2DProt project [3], a free software suite which is probably working but not (yet) at the simplicity level of commercial offers - IMAL [3] seems to be a general math package working with 2D gels Some software companies (I'm sure for Progenesis/Nonlinear) are offering you the software with temporary keys (so you pay less money but only for a Otherwise, you can find on the web companies that are processing gels for you and doing the analysis too (of course, you have to electronically send them your gel images and pay a fee). Hope this helps, Jean-Etienne [1] 2D Gel Analysis Software: http://en.wikipedia.org/wiki/Two-dimensional_gel_electrophoresis#2D_Gel_Analysis_Software [2] Gellab: http://www.lecb.ncifcrf.gov/gellab/ [3] IMAL: http://brneurosci.org/imal.html -- Jean-Etienne Poirrier http://www.poirrier.be/~jean-etienne/ http://www.epot.org/blog/ From allicindixvin from gmail.com Mon Jan 14 09:17:39 2008 From: allicindixvin from gmail.com (kyoli) Date: Mon Jan 14 12:56:09 2008 Subject: [Protein-analysis] protein extraction Message-ID: <0d637293-9504-41a1-a0e7-fdc9a32bff3d@d21g2000prf.googlegroups.com> i am performing 1-D gel electrophoresis for protein from leave but chlorophyll is inhibiting for which i used acetone with tris. do anyone have any method to extract protein in this combination without using liquid nitrogen From shettylv from gmail.com Wed Jan 16 00:17:57 2008 From: shettylv from gmail.com (leelavati shetty) Date: Wed Jan 16 12:22:45 2008 Subject: [Protein-analysis] USP silver staining method help Message-ID: <7683b8c40801152117xd471cbbw6c40817613f24cb9@mail.gmail.com> Hi Everyone, We are using 13% Resolving and 4% stacking gel and tris glycine SDS electrohoresis buffer with discontinuous gel system. We had tried out the silver staining technique mentioned in the EP / USP 30 pharmacopoeia since we have to comply pharmacopoeia requirements for our protein. But the gel darkens and method is not very sensitive. Is there something wrong in the procedure we are following. Can anyone tell me exact procedure to perform silver stg as per USP and precautions to be taken. Thanks Exec -- Leelavati Shetty From engelbert_buxbaum from hotmail.com Wed Jan 16 13:00:14 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Wed Jan 16 15:17:50 2008 Subject: [Protein-analysis] Re: 2D gel lossing high molecular weights References: Message-ID: Am 22.10.2007, 12:17 Uhr, schrieb Isabelle Holdridge : > I have been running 2D gels for soy bean total soluble proteins and I am > consistently losing the higher molecular weight proteins. These same > proteins are visible on the 1D gels. I have tried a variety of changes > in my protocol, such as, changes with the running buffers, IPG buffers > and detergents, and ph gradients, but all to no avail. The lower > molecular weights on the 2D gel are visible and appear to be nicely > focused. I am not getting much background staining or streaking. Any > suggestion would be appreciated. With so little info it is difficult to guess. What is the MW of the proteins you are looking for? With acrylamide down to 5% 200 kDa is about the maximum, beyond that you need to strengthen your gels with agar as they get to soft. How are you doing your first dimension run? I have never been able to get ampholine tube gels to work properly, too much protein precipitates near the starting zone. However, IPG gels work fine even with large membrane proteins like Mdr1 (160 kDa). I use the sample solution to rehydrate the gel (8 h at 50 V, 18 deg C), giving me a reasonable sample volume, I also found you need about 25 kV*h to focuss a 7 cm gel, about twice of what the manual said. Note also that proteins with extreme pI are lost irrespective of MW. Before the 2nd dimension run, the IEF gel must be equilibrated with sample buffer (10 min at 37 deg C in a sealed tube on an end-over-end mixer). This allows the detergent (SDS in most cases) to bind to the proteins in the gel. Then mount the gel with stacking gel buffer containing 0.5% agarose. From engelbert_buxbaum from hotmail.com Wed Jan 16 13:04:11 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Wed Jan 16 15:18:04 2008 Subject: [Protein-analysis] Re: Problems with silver staining References: Message-ID: Am 29.10.2007, 05:15 Uhr, schrieb terence diane fabella : > conversation, that you could give Matt a copy of the protocol on how to > make a silver stain, if he likes. Can I ask a copy of it too? Heukeshoven & Dernick: Electrophoresis 9 (1988) 28-32 works well. You do not need the different temperatures, just working at 37 deg C for all steps is fine. From engelbert_buxbaum from hotmail.com Wed Jan 16 13:14:34 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Wed Jan 16 15:18:09 2008 Subject: [Protein-analysis] Re: picomolar Kd or Ki References: Message-ID: Am 06.12.2007, 14:20 Uhr, schrieb Ligong Wang : > Can one get a Kd (or Ki) value from an assay that is lower than the > amount (concentration) of protein/enzyme used therein? The answer from Radio Eriwan: Yes in principle, but the error increases. Best results are obtained when both [E] and [S] are within 1 order of magnitude of Kd. But the law of mass action does apply outside that range too. For a good discussion on practical enzyme kinetics see the book by Bisswanger (Enzyme Kinetics - Theory and Methods), but the next english edition is about to be published this spring, so do not buy just yet (those who can read German may of course get the original, which was publihed some time ago). From engelbert_buxbaum from hotmail.com Wed Jan 16 13:41:37 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Wed Jan 16 15:18:28 2008 Subject: [Protein-analysis] Re: protein concentration in Ethanol References: Message-ID: Am 19.12.2007, 16:58 Uhr, schrieb Fery : > I have made a lipid extract from pollen by folch method and dissolve the > my lipids in pure Ethanol. I want to measure the percent of protein > contamination in my extract. please let me know what is the best method > for measurment of protein in ethanol. Theoretically there shouldn't be any, since proteins are not supposed to be alcohol soluble (and neither in chloroform used during the extraction). Short, hydrophobic peptides perhaps. Their determination would not be easy however, because they could not be separated from the lipid by Alcohol/Chloroform or by TCA/DOC, and probably not with phenol either. Biuret (and its congeners like Lowry or BCA) may not work if the peptides are very short, dye binding neither and aromatic amino acids for A280 may not be present. If I suspected that such peptides were present, I would probably try a 2D TLC with Kieselgel G plates (Merck, activated 1 h at 130 deg C and allowed to cool protected from humidity) with 1-butanol/acetic acid/water 6+2+2 as the first and chloroform/methanol/ammonia 50+40+5 as 2nd dimension mobile phase. Detect lipids by incubating the plates in a closed chromatography tank with a few crystals of iodine. Mark the spots with a pencil as the colour is not permanent. Then spray with 1 mg/ml ninhydrine in water saturated n-butanol and heat for 10 min at 85 deg C. Any purple spots except PS and PE would warrant further investigation. From engelbert_buxbaum from hotmail.com Wed Jan 16 13:48:09 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Wed Jan 16 15:18:32 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: Am 04.01.2008, 16:03 Uhr, schrieb DK : > Well, that means you need to get rid of the medium and all of my > suggestions are potentially applicable. Yes, I think so too. I'd probably try to concentrate my extract in an Amicon pressure cell, add a suitable buffer and repeat a couple of times. That should remove most of the low MW contaminants. Even better of course to pellet the cells in a centrifuge, resuspend and then lyse in a defined medium. I never liked the idea of proprietary reagents where you don't know what the hell you are getting into. From engelbert_buxbaum from hotmail.com Wed Jan 16 14:14:01 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Wed Jan 16 15:18:36 2008 Subject: [Protein-analysis] Re: Western blot using FACS cells References: Message-ID: Am 03.01.2008, 13:19 Uhr, schrieb Melissa Mazan : > I am trying to identify proteins using WB on cells that have already > been through FACs.I'm not having much luck with samples that have less > than a million >cells. Any suggestions? A mammalian cell has about 10 um diameter (1.2e-9 cm3 volume or about 1e-9 g weight given that the density of cytosol is slightly above 1). Protein content about 10% by weight = 1e-10 g of protein. Thus your 1e6 cells would contain approx 100 ug of total protein, which is a reasonable to smallish load for a minigel. Distribute that over, what, 30 bands and you have just enough protein to detect an average band with Coomassie. Those with less than average amounts of protein perhaps with silver. Smaller gels (e.g. PhastSystem) may give you a slightly higher sensitivity, but miracles you should not expect. From engelbert_buxbaum from hotmail.com Wed Jan 16 14:18:19 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Wed Jan 16 15:18:40 2008 Subject: [Protein-analysis] Re: problem of concentrating protein References: Message-ID: Am 10.01.2008, 01:01 Uhr, schrieb : > The protein I am purifying is a kind of zinc finger protein which > has 81 amino acid and its theoretical pI is 9.19.But after purified > with Hitrap Heparin column the target protein were still stable,then > when I use the binding buffer(20mM Tris,10mM Mer,100mM NaCl and 1mM > ZnCl2,pH 7.0)to dialyze in order to remove the 2M NaCl which were used > in eluting the protein from the Heparin column, I found that there are > many sediment in it. > How to solve this problem?Is there any buffer which is more siutable > than Tris? Since at pH7.0 Tris is not as efficient as it was. > Plus I tried phosphate buffer,but it will interact with the ion > zinc (within zinc finger protein) and produce sendiment too. What is the composition of your elution buffer? Does it contain Zn? Does the precipitation also occur when the binding buffer does not contain Zn? From engelbert_buxbaum from hotmail.com Wed Jan 16 14:23:04 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Wed Jan 16 15:18:45 2008 Subject: [Protein-analysis] Re: 2-D analysis software References: Message-ID: Am 12.01.2008, 08:05 Uhr, schrieb : > Hallo, > > > Anybody out there with an idear of software available over the internet > that may help us do some analysis of differentially expressed proteins > through 2-D? We have tried "Flicker" unfortunately it seems the format in > which we have saved our photos is not compatible. We would really > appreciate any suggestion. Get IrfanView (http://www.irfanview.net/) and convert the image into a format that is compatible with your software. Gimp may also do the same, but is somewhat less easy to use. From engelbert_buxbaum from hotmail.com Wed Jan 16 14:25:56 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Wed Jan 16 15:18:49 2008 Subject: [Protein-analysis] Re: protein extraction References: <0d637293-9504-41a1-a0e7-fdc9a32bff3d@d21g2000prf.googlegroups.com> Message-ID: Am 14.01.2008, 10:17 Uhr, schrieb kyoli : > i am performing 1-D gel electrophoresis for protein from leave but > chlorophyll is inhibiting for which i used acetone with tris. do > anyone have any method to extract protein in this combination without > using liquid nitrogen Chloroform/Methanol precipitation should work @article{Wes-84, AUTHOR= {D. Wessel and U.I. Fl?gge}, TITLE= {A Method for the quantitative recovery of protein in the presence of detergents and lipids}, JOURNAL= {Anal. Biochem.}, VOLUME= {138}, YEAR= {1984}, PAGES= {141-143}, LANGUAGE= {engl} } From proteininteraction from gmail.com Thu Jan 17 04:31:30 2008 From: proteininteraction from gmail.com (Simon) Date: Thu Jan 17 09:57:10 2008 Subject: [Protein-analysis] CALL FOR CHAPTERS on Biological Data Mining in Protein Interaction Networks Message-ID: Biological Data Mining in Protein Interaction Networks A book edited by X.-L. Li and S.-K. Ng, Institute for Infocomm Research, Singapore Introduction The cellular machinery is a complex system with a multitude of bio- molecular interactions. Most of the cellular processes are mediated by protein-protein interactions (PPIs). Recently, high-throughput methods for detecting PPIs have revealed global pictures of protein interactions on a genomic scale, making it possible to interrogate the interplay of the bio-molecules in carrying out critical life processes at the networked co-operative level. As a result, modern biology's focus has shifted from scrutinizing single molecules to analyzing large complex networks. The Overall Objective of the Book Genome-wide screenings of PPIs can be represented as large protein interaction networks (PINs) in which the nodes represent individual proteins, and the links represent the existence of biological interactions between the corresponding pairs of proteins. In this book, we will present bioinformatics methods that are designed for the computational analyses of the PINs to better understand critical life processes such as the cellular signaling, molecular trafficking, and gene regulatory pathways. Each of the chapters will serve as a tutorial for network-based computational analysis PINs. The objective of this book is to disseminate the research results and best practice from researchers and practitioners working on bioinformatics, data mining, and proteomics. We aim to present the various methodologies in an accessible way to our inter-disciplinary audience, through which we hope to bring better awareness of this interesting and c! hallenging problem to inspire new solutions and applications. The Target Audience The book is aimed at three overlapping audiences: (1) Researchers in the areas of bioinformatics, data mining, machine learning and data structure; (2) Practitioners in the industry in these areas; (3) Instructors and postgraduate students in colleges and universities. This book will be an essential resource for professionals and researchers who wish to learn about the application of advanced data mining techniques in protein interaction networks. In addition, it can be used as a reference for Masters and Ph.D. students studying bioinformatics. Recommended topics include, but are not limited to, the following: Understanding the Properties of a Protein Interaction Network Protein Interactions: Importance and Detection Protein Interactions: In Silico Prediction Interacting Domain Prediction and Usage Interacting Motif: Prediction and Usage Lethal Proteins: Roles and Identification Protein Function: Classification and Annotation Protein Complexes: Construction via Computational Means Functional Module: Detection and Biological Significance Network Motif: Detection and Biological Relevance Network Cleansing: Reliable Interaction Network Conserved Network: Data Mining Across Species PPI and Diseases: Mining Disease Genes from Protein Interaction Networks Computational Analysis for Cell Signaling Computational Analysis for Molecular Trafficking Computational Analysis for Receptor Applications Integrating Protein Interaction Network with Other Biological Resources Submission Procedure Researchers and practitioners are invited to submit on or before March 31, 2008, a 2-5 page manuscript proposal clearly explaining the mission and concerns of the proposed chapter. Authors of accepted proposals will be notified by April 10, 2008 about the status of their proposals and sent chapter organizational guidelines. Full chapters are expected to be submitted by May 31, 2008. All submitted chapters will be reviewed on a double-blind review basis. The book is scheduled to be published by IGI Global, www.igi-global.com, publisher of the IGI Publishing (formerly Idea Group Publishing), Information Science Publishing, IRM Press, CyberTech Publishing, Information Science Reference (formerly Idea Group Reference), and Medical Information Science Reference imprints. Inquiries and submissions can be forwarded electronically (Word document) or by mail to: Dr. Xiao-Li Li Data Mining Department Institute for Infocomm Research 21 Heng Mui Keng Terrace, Singapore 119613 Tel: 65 68748452 * Fax: 65 67768109 E-mail: xlli@i2r.a-star.edu.sg Website: http://www1.i2r.a-star.edu.sg/~xlli/PPI_BOOK.html From gormicon from gmail.com Fri Jan 18 07:14:47 2008 From: gormicon from gmail.com (GormEnOrm) Date: Fri Jan 18 08:53:21 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: <72708a12-8ea8-406f-af3f-0e6c0bf5b442@u10g2000prn.googlegroups.com> On 3 Jan., 06:44, 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. > > Thanks! Hi Tiwari, I have had the same problem with Baculovirus System. Media contains both a chelating agent and a imidazole-like molecule (probable histidine in high amounts) :-(. For a long time we have dialysed samples for three days against 5 x 8 L buffer. Such a waste of water, time and chemicals. But there are smart solutions. You could try ion-exchange column to concentrate protein and get rid of oppositely charged contaminants. Then the sample volume would be lowered to 10 - 20 mL, which can be dialysed or desalted otherwise. If you purify an enzyme and have an assay for detection, this can quickly be optimized. The best solution is a CrossFlow machine. We just bought one in my lab. It takes in fx. 1-600 mL sample (we only have 600 mL as max, but machine can take more), concentrates to 32 mL and diafiltrates (10 kDa cut-off) with 160 mL buffer. This results in approx. 45 mL concentrated sample with a 95 % recovery of protein and a 95-98 % reduction in small contaminants, just ready to go to the Ni-NTA column. All done in 3 - 4 hrs. Nothing can beat that. - Regards Gormicon From sandy_zhu_2006 from hotmail.com Sat Jan 19 07:29:04 2008 From: sandy_zhu_2006 from hotmail.com (sandy_zhu_2006@hotmail.com) Date: Sat Jan 19 14:10:59 2008 Subject: [Protein-analysis] Re: problem of concentrating protein References: Message-ID: <778fee5c-a2b5-4d01-83e5-1148e5baf1d2@i29g2000prf.googlegroups.com> On 1=D4=C217=C8=D5, =C9=CF=CE=E73=CA=B118=B7=D6, "Dr Engelbert Buxbaum" wrote: > Am 10.01.2008, 01:01 Uhr, schrieb : > > > The protein I am purifying is a kind of zinc finger protein which > > has 81 amino acid and its theoretical pI is 9.19.But after purified > > with Hitrap Heparin column the target protein were still stable,then > > when I use the binding buffer(20mM Tris,10mM Mer,100mM NaCl and 1mM > > ZnCl2,pH 7.0)to dialyze in order to remove the 2M NaCl which were used > > in eluting the protein from the Heparin column, I found that there are > > many sediment in it. > > How to solve this problem?Is there any buffer which is more siutable > > than Tris? Since at pH7.0 Tris is not as efficient as it was. > > Plus I tried phosphate buffer,but it will interact with the ion > > zinc (within zinc finger protein) and produce sendiment too. > > What is the composition of your elution buffer? Does it contain Zn? Does = > the precipitation also occur when the binding buffer does not contain Zn? My elution buffer do have Zn(1mM ZnCl2) in it.How to improve its solubleness is the mayor concern right now. From clement from bio.mls.eng.osaka-u.ac.jp Sun Jan 20 20:54:39 2008 From: clement from bio.mls.eng.osaka-u.ac.jp (Clement Angkawidjaja) Date: Sun Jan 20 22:22:46 2008 Subject: [Protein-analysis] Re: Proteins Digest, Vol 32, Issue 11 References: <200801201703.m0KH3AL21935@net.bio.net> Message-ID: <000801c85bd0$9642bb20$3100a8c0@CLEMENT> I purified a nuclear receptor once and it requires high concentration of salt (>500 mM) to remain soluble. Other osmolytes (glycerol, non-ionic detergents) can replace salt for nuclear receptors (confirmed). I also heard (although have not tried it) that macromolecular crowding agents (PEG20K, ficol70) can also increase solubility. The point is, many intracellular proteins, especially DNA-interacting ones stay stable in a "crowded" environment (the cytosol and nucleoplasma are crowded). The selection of crowding agents will depend on what experiments you will do with your purified proteins. Clement ----- Original Message ----- From: To: Sent: Monday, January 21, 2008 2:03 AM Subject: Proteins Digest, Vol 32, Issue 11 > 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. Re: problem of concentrating protein (sandy_zhu_2006@hotmail.com) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Sat, 19 Jan 2008 04:29:04 -0800 (PST) > From: sandy_zhu_2006@hotmail.com > Subject: [Protein-analysis] Re: problem of concentrating protein > To: proteins@net.bio.net > Message-ID: > <778fee5c-a2b5-4d01-83e5-1148e5baf1d2@i29g2000prf.googlegroups.com> > Content-Type: text/plain; charset=GB2312 > > On 1??17??, ????3??18??, "Dr Engelbert Buxbaum" > wrote: >> Am 10.01.2008, 01:01 Uhr, schrieb : >> >> > The protein I am purifying is a kind of zinc finger protein which >> > has 81 amino acid and its theoretical pI is 9.19.But after purified >> > with Hitrap Heparin column the target protein were still stable,then >> > when I use the binding buffer(20mM Tris,10mM Mer,100mM NaCl and 1mM >> > ZnCl2,pH 7.0)to dialyze in order to remove the 2M NaCl which were used >> > in eluting the protein from the Heparin column, I found that there are >> > many sediment in it. >> > How to solve this problem?Is there any buffer which is more siutable >> > than Tris? Since at pH7.0 Tris is not as efficient as it was. >> > Plus I tried phosphate buffer,but it will interact with the ion >> > zinc (within zinc finger protein) and produce sendiment too. >> >> What is the composition of your elution buffer? Does it contain Zn? Does >> the precipitation also occur when the binding buffer does not contain Zn? > > My elution buffer do have Zn(1mM ZnCl2) in it.How to improve its > solubleness > is the mayor concern right now. > > > ------------------------------ > > _______________________________________________ > Proteins mailing list > Proteins@net.bio.net > http://www.bio.net/biomail/listinfo/proteins > > End of Proteins Digest, Vol 32, Issue 11 > **************************************** From sanjaysingh765 from gmail.com Tue Jan 22 04:15:26 2008 From: sanjaysingh765 from gmail.com (chunnu) Date: Tue Jan 22 12:23:24 2008 Subject: [Protein-analysis] quiery Message-ID: <3cb9dd9b-d577-42dc-8f36-5898cd95b017@s12g2000prg.googlegroups.com> Hi There, I m trying 2 build a theoretical of a protein but unfortunately the template 4 whole protein is not availabe in PDB so i build 3/4 th part of protein by compertive modelling while remaining by de novo method..now I want to ligate the backbone of the both model in order to generate a complete model.may I do it??if yes then how..plz suggest a proper method..my mail ID is sanjaysingh765@gmail.com regards sanjay From brighttomorrow from gmail.com Wed Jan 23 21:37:15 2008 From: brighttomorrow from gmail.com (Nico) Date: Wed Jan 23 23:18:52 2008 Subject: [Protein-analysis] Re: His-tagged protein purification from yeast (k lactis) supernatant Message-ID: <200801232137150469137@gmail.com> Hi, Gormicon, Do you happen to know which brand and how much of the CrossFlow machine. Many thanks, Nico From allicindixvin from gmail.com Wed Jan 30 04:36:06 2008 From: allicindixvin from gmail.com (kyoli) Date: Wed Jan 30 15:30:08 2008 Subject: [Protein-analysis] query Message-ID: hi, i need standard of thymoquinone, a volatile component of nigella species. do anyone have idea from where will i get it From boconnie from gmail.com Thu Jan 31 23:43:19 2008 From: boconnie from gmail.com (Connie Lam) Date: Fri Feb 1 15:48:51 2008 Subject: [Protein-analysis] Requesting protocol for human CD38 western analysis In-Reply-To: References: Message-ID: <93f327860801312043q76210882xba9eb6ad9c5b0512@mail.gmail.com> 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