help

BMKAO at CCVAX.SINICA.EDU.TW BMKAO at CCVAX.SINICA.EDU.TW
Fri Oct 29 19:15:00 EST 1993


Dear receiver:
    I have some problems about GAP( GTPase activating protein) assay, and 
need your help to resolve them.
    The GAP assay system I use is described as the following text. Small 
molecular weight G protein( smg) was pre-formed GTP-bound form and pass 
through desalt spin column to remove excess free GTP, added into reaction 
solution containing cell lysate, incubated for proper time at RT and 
stopped by SDS/EGTA buffer. The mixture was applied to a TLC plate to 
obtain GTP/GDP for estimation of single turnover rate.
    According to previous reports, smg  only conveted GTP to GDP in presence of 
cell lysate or without cell lysate. Except GTP and GDP,  GMP was found 
on the TLC plate when cell lysate was added ( not found in smg alone). I re
-purified the smg-GTP complex to rule out the nucleotide kinase or 
phosphatase effects. Can I ignore GMP in calculation GTP/(GTP+GDP)? By 
using this method I can not get more accurate result of enhancement of Kcat 
induced by cell lysate. Is GAP too fragile to obtain reproducible result?
The experiment was performed at RT without water bath control. Is it 
necessary to perform experiments under such as critical condition? If I 
want to compare GAP activity of different stimulated cell lysates, how 
should I do to obtain different stimulated lysates?
    If you know how to resolve these problems, please let me know. Thanks 
a lot.						
						Lin, Chong-Gee 10/29/'93
   						




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