From drmuhanad1976 from yahoo.com Mon Mar 3 04:57:41 2008 From: drmuhanad1976 from yahoo.com (Muhanad El-Etibi) Date: Mon Mar 3 13:39:16 2008 Subject: [Protein-analysis] Colleague request your help Message-ID: <468781.22753.qm@web56911.mail.re3.yahoo.com> Dear Sir This is Dr. Muhanad M. Ahmed Abed (Ph.D. Microbiology/Immunology) I try to download the wingene/winpep software using the link below: http://www.ipw.agrl.ethz.ch/~lhennig/winpep.html however, I kept fail. Please if you know another link I'll appreciate if you can send it to me. Thank you very much in advance for your time and consideration. Sincerly your Dr. Muhanad M. AHMED ABED Head of Dep. Of Clinical Lab. Science College of Pharmacy/Kerbala Univ Kerebala/Iraq --------------------------------- Looking for last minute shopping deals? Find them fast with Yahoo! Search. From tfcheng from gmail.com Sat Mar 8 16:01:28 2008 From: tfcheng from gmail.com (Tsu-Fan Cheng) Date: Sat Mar 8 20:43:56 2008 Subject: [Protein-analysis] PEI and ammonium sulfate ppt Message-ID: Hi, I am purifying a protein with a protocol that I used last time to purify a similar protein. This protocol involves PEI (polyethyleneimine) fractionation and ammonium sulfate precipitation. The idea of AS ppt is to remove residual PEI from the solution. However, this time the PEI is still there and my protein precipitated. The only difference I can tell from this exp to the last one is that I dissolve the AS ppt pellete with a solution containing 250mM NaCl, which is none in the previous exp. I thought the [NaCl] won't matter since PEI should be removed after AS ppt. I wonder if someone can explain PEI+AS procedure to me so I have a better understanding and control over this protocol in the future. thank you. From engelbert_buxbaum from hotmail.com Mon Mar 10 09:09:34 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Mon Mar 10 11:42:48 2008 Subject: [Protein-analysis] Re: PEI and ammonium sulfate ppt References: Message-ID: Am 08.03.2008, 17:01 Uhr, schrieb Tsu-Fan Cheng : > The idea of AS ppt is to remove residual PEI from the solution. > However, this time the PEI is still there and my protein precipitated. Almost any protein can be precipitated with AS, that is just a question of concentration. Some proteins already precipitate at 20% satturation, others only at 80%. In this case you apparently have a protein that precipitates at relatively low conc of AS, before the PEI precipitates. That's fine, just spin down the protein, remove the supernatant with the PEI, wash the precipitate with AS solution of the same conc as used for pelleting, spin again, discard the supernatant and redissolve your protein in a buffer of your choice. Depending on what you want to do you may have to remove the remaining AS by either dialysis or gel filtration (''desalting column''). Note also: - many proteins store very well as AS precipitate in mother liquor at 4 deg C. - Add the AS slowly and in the cold. Stop the addition when just a slight haze develops in the protein solution, than keep o/n in the fridge. That way you get best separation between your protein and whatever remains in solution. - Any book on protein purification will discuss this in more detail. From ikramaujla from hotmail.com Wed Mar 12 01:11:53 2008 From: ikramaujla from hotmail.com (Muhammad Ikram Aujla) Date: Wed Mar 12 12:44:50 2008 Subject: [Protein-analysis] Proteins estimation Message-ID: Dear serita did you get the method for protein estimation? can you kindly share with me as well? ************************** Muhammad Ikram Aujla Assistant Professor, Department of Chemistry, Forman Christian College ( A Chartered University), Lahore - 54600, Pakistan. Mobile # + 92 (0) 333 4066900 Mail To : ikramaujla@hotmail.com _________________________________________________________________ Express yourself instantly with MSN Messenger! Download today it's FREE! http://messenger.msn.click-url.com/go/onm00200471ave/direct/01/ From yogeshasd from yahoo.co.in Thu Mar 20 23:51:42 2008 From: yogeshasd from yahoo.co.in (S. D. Yogesha) Date: Sat Mar 22 06:07:11 2008 Subject: [Protein-analysis] refolding problem Message-ID: <207342.71140.qm@web94601.mail.in2.yahoo.com> Hi everyone, My protein has c terminal His tag. When I express it more than half remains in IBs. Even after using more culture volume is not helping to get enough protein for work. [What ever little I get is aggregating almost near to 100%. I want to address both problem but one after the other. I know this aggregation is because I mutated 5 glutamine and 1 lysine residue as aa patch (EEE and EKE).} Trying different temperature, strains and induction method did not help me increasing solubilization. I have not tried cheperons. I tried to solubilize the IBs with 8M urea as in standard protocol, was surprised to find that protein is not binding to both Nickle and Talon column. I asked a colleague and he said His6 and His7 had never given him problem. My tag is His8 and I did not get any reason why my protein is not binding in denatured condition when I can purify same in native condition. Can any one educate me if I am missing something in understanding this. I followed J Struct Funct Genomics. 2005;6(2-3):177-82, and instead of beta cyclodextrin I used 2 hydroxyl beta cyclodextrin. Entire protein was in flowthrough and first wash even after overnight binding. Thanks Yogi ____________________________________________________________________________________ Looking for last minute shopping deals? Find them fast with Yahoo! Search. http://tools.search.yahoo.com/newsearch/category.php?category=shopping From j.jaehnichen from tu-bs.de Tue Mar 25 05:23:25 2008 From: j.jaehnichen from tu-bs.de (Sunlacrimula) Date: Tue Mar 25 11:36:02 2008 Subject: [Protein-analysis] protein mw Message-ID: <85305f1f-0d00-4f06-959d-a8cbf31cee94@d45g2000hsc.googlegroups.com> Hi, I am working with an unknown protein. The calculated size should be about 70 kDa. I produce it in SaOS-Cells and it is secreted into the culture medium. It has a his and a myc tag. When I analyse it on SDS-Page and Western Blot I always get a big and clear band at about 100 kDa and only a very thin band at 70 kDa. I know that inside the cell it has the right size but after the secretion it is 30 kDa larger. I already had it in MS (tryptic digestion) and only my protein and no other protein came up. So what can it be that is bound to my protein? We already discussed glycosylation. But people told me for that the band is to clear. We checked with a glycosylation inhibitor and the protein is only about 5 kDa smaller. If you have any idea what I could check for, that would be great. Thank you very much. From markII from came.sbg.ac.at Tue Mar 25 09:39:25 2008 From: markII from came.sbg.ac.at (Markus Wiederstein) Date: Tue Mar 25 12:36:46 2008 Subject: [Protein-analysis] TopMatch protein structure alignment and superposition web service Message-ID: We are pleased to announce the release of TopMatch-web, a public web service for the alignment and superposition of protein structures and the instant visualization of structural similiarities. We believe that TopMatch is an exceptionally effective, accurate, and enjoyable program for the investigation of protein structure similarities. For further information and instructive examples see Sippl & Wiederstein (2008) A Note on Difficult Structure Alignment Problems. Bioinformatics 24, pp. 426-427 TopMatch-web is available as a public web service at http://topmatch.services.came.sbg.ac.at. Enjoy! The TopMatch Team Center of Applied Molecular Engineering University of Salzburg Austria From npakentieva from vcu.edu Mon Mar 31 15:11:05 2008 From: npakentieva from vcu.edu (Natalia P Akentieva/FS/VCU) Date: Tue Apr 1 13:36:26 2008 Subject: [Protein-analysis] extraction proteins from gel Message-ID: Hello, I am interesting about passive extract= ion proteins from the gels, how to do it? Could you send me a references fo= r this approach and what yield of protein can I get it? Best regards, Natalia Akentieva