From owner-7tms_r@hgmp.mrc.ac.uk Mon Dec 20 20:12:52 1999 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id D600A17AF7; Mon, 20 Dec 1999 20:12:50 +0000 (GMT) Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 1) id 5F25D17AF4; Mon, 20 Dec 1999 20:12:49 +0000 (GMT) To: nobody@net.bio.net X-Really-To: 7tms_r@net.bio.net Newsgroups: bionet.molbio.proteins.7tms_r Date: Fri, 24 Sep 1999 20:00:02 -0400 From: damania Subject: Help-Endocytosis!!!! Message-Id: <19991220201249.5F25D17AF4@mercury.hgmp.mrc.ac.uk> Sender: owner-7tms_r@hgmp.mrc.ac.uk Precedence: bulk --- Note from moderator --- This newsgroup has been non-functional due to issues at BIOnet. This is a test message. --------- Hi All !! I am currently trying to measure the rate of endocytosis of my receptor protein. This is the way I do it: 1) Bind labeled antibody to surface of cells for 30 mins @ 4C 2) Wash off excess unbound antibody and take aliquot for total antibody quantitation 3) Incubate cells at 37 C for different time intervals 4)Take aliquots 5) Wash cells with 150 mM glycine + PBS (pH =2) to remove any antibody bound to surface of cells. (ACID WASH STEP) 6) Measure the amount of internalized antibody. HOWEVER, MY PROBLEM IS THAT AFTER MY ACID WASH STEP MY CELLS ARE ALL DEAD !!!! Can anyone help me out?? what am i doing wrong???? Thanks in advance damania From owner-7tms_r@hgmp.mrc.ac.uk Mon Dec 20 22:22:52 1999 Return-Path: Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 110) id 17AF417B12; Mon, 20 Dec 1999 22:22:51 +0000 (GMT) Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 1) id 9EFE917B0B; Mon, 20 Dec 1999 22:22:49 +0000 (GMT) To: nobody@net.bio.net X-Really-To: 7tms_r@net.bio.net Newsgroups: bionet.molbio.proteins.7tms_r From: rm1950@rocketmail.com Subject: Re: Help-Endocytosis!!!! Date: 20 Dec 1999 22:11:59 -0000 Organization: The University of Melbourne References: <83m2k0$3ou$1@mercury.hgmp.mrc.ac.uk> Message-Id: <19991220222249.9EFE917B0B@mercury.hgmp.mrc.ac.uk> Sender: owner-7tms_r@hgmp.mrc.ac.uk Precedence: bulk Does it really matter if the cells are dead? If all you want to know is the rate of internalization, then you'll have this information, whether the cells are viable or not. If you want to maintain viable cells to carry on into culture, then you may have to play with detergents and/or denaturants to get rid of excess antibody. Not much help maybe...but what do you actually want to achieve? In article <83m2k0$3ou$1@mercury.hgmp.mrc.ac.uk>, damania wrote: >I am currently trying to measure the rate of endocytosis >of my receptor protein. This is the way I do it: > >1) Bind labeled antibody to surface of cells for 30 mins @ 4C >2) Wash off excess unbound antibody and take aliquot for total antibody >quantitation >3) Incubate cells at 37 C for different time intervals >4)Take aliquots >5) Wash cells with 150 mM glycine + PBS (pH =2) to remove any >antibody bound to surface of cells. (ACID WASH STEP) >6) Measure the amount of internalized antibody. > >HOWEVER, MY PROBLEM IS THAT AFTER MY ACID WASH >STEP MY CELLS ARE ALL DEAD !!!! > >Can anyone help me out?? >what am i doing wrong???? > >Thanks in advance >damania - ---