Subject: help

From: [email-masked]
Date: 17 Nov 93 00:39:00 GMT

I have some problems in GAP( Gtpase activating protein) assay. First, I 
describe the assay system  that I use for GAP. Smg( small G protein) was 
pre-formed GTP form, add the GTP form smg into cell lysate and incubate for 
proper time, the reaction was stopped by adding SDS/EDTA solution, the 
stopped mixture was developed by TLC and obtain GTP/GTP+GDP ratio as GTPase 
activity. Because I have no smg antibody, I discarded the 
immunoprecipitation step before stop reaction.
Problem 1. Is immunoprecipitation a essential step? Because 
immunoprecipitation takes time and seemed to affect estimation of GTPase 
activity. Another problem that I concerned is that immunoprecipitation 
cause to get lower lower radioactivity because some guanosine nucleotide 
exchange factor cause radioactive bound-form guanosine nucleotides turned 
into free form because of replacement of cold free guanosine nuceleotides.
Problem 2. If I want to measure GAP activity change during stimulation( 
such as high potassium), how can I obtain the stimulated cell lysates at 
different time points?
    If you know how to resolve these problems, please tell me. Thank you!
						Lin, Chong-Gee