From owner-7tms_r@hgmp.mrc.ac.uk Wed May 24 16:05:15 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 1066B17B0D; Wed, 24 May 2000 16:05:12 +0100 (BST) Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 60001) id 308D017B51; Wed, 24 May 2000 16:05:11 +0100 (BST) To: 7tms_r@net.bio.net Newsgroups: bionet.molbio.proteins.7tms_r From: nestgrp@world.std.com (Amos Heckendorf) Subject: IMAC tips for phophopeptides or not? Date: 19 May 2000 14:41:36 +0100 Organization: Software Tool & Die, Brookline MA Message-Id: <20000524150511.308D017B51@mercury.hgmp.mrc.ac.uk> Sender: owner-7tms_r@hgmp.mrc.ac.uk Precedence: bulk Subject: IMAC tips for phosphopeptides or not? Dear 7tms_r newsgroup members: As you are aware, there are several vendors for microSPE tips and spin columns (ZipTip(tm), SuproTip(tm), TipColumns, MiniSpin(tm), etc.). I present this here for the purposes of discussion only. We will all hopefully benefit from a open sharing of information. I would like to suggest that in the face of overwhelming interest in IMAC for phosphopeptide isolation for protein digest work, IMAC as the sole microSPE clean-up tool is not the best sole choice. As many of you have found out, the SDS in the gels poisons the IMAC surface. Yes, you can wash out the dyes and SDS prior to IMAC binding, but there remains a strong possibility that you may not have taken out enough to leave sufficient binding capacity in the columns. The reason that this is an issue is that in tip column microSPE, there is much less packing material to work with and you reach the binding capacity limits more easily than in a spin column or larger devices. I suggest, in the face of your enthusiasm, that you consider instead the use of HILIC, as Jen=F6 et al. (Anal. Biochem. 215 (1993) 292) have show= n for the removal of SDS and salts from electroeluted proteins. The SDS and dyes do not bind under the loading conditions and your phosphopeptides will be the last to come out, since they bind more strongly than less polar molecules. Then, if you want to sub fractionate these polar products, you can pick out the phosphopeptides from this fraction with an IMAC tip with better assurances of success. Once the IMAC is contaminated, it is dead. While not completely foolproof, this seems better than the use of C18. With C18 materials the SDS is retained longer than the peptides, but not that much longer. If too many bed volumes of liquid are used, or too much organic is used (ca. 45-65% MeCN), the SDS will elute along with the peptides and contaminate the IMAC surface. I hope this helps refine your thinking and I welcome input from the group as to how you are solving this application. Sincerely, Amos Heckendorf The Nest Group For examples on HILIC and microSPE protocols, see: http://world.std.com/~nestgrp/pdf/pdf.html - --- ------- End of forwarded message ------- From owner-7tms_r@hgmp.mrc.ac.uk Wed May 24 16:05:25 2000 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 2EEAE17B0D; Wed, 24 May 2000 16:05:24 +0100 (BST) Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 60001) id 0B8DC17B4F; Wed, 24 May 2000 16:05:22 +0100 (BST) To: 7tms_r@net.bio.net Newsgroups: bionet.molbio.proteins.7tms_r From: elisabeth.falkenstein@kpha.ma.uni-heidelberg.de ("Falkenstein, Elisabeth") Subject: expression library enriched in GPCRs Date: 24 May 2000 09:49:33 +0100 Organization: BIOSCI/MRC Human Genome Mapping Project Resource Centre Message-Id: <20000524150522.0B8DC17B4F@mercury.hgmp.mrc.ac.uk> Sender: owner-7tms_r@hgmp.mrc.ac.uk Precedence: bulk Hi, Our group is working on steroid hormone action. Does anyone know a person or group which have cDNA expression libraries which are enriched in GPCR. I am especially interested in (putative) pheromone receptors (mammalian). Of course we are interested in an official cooperation. Thanks E. Falkenstein - ---