Ras activity

Lani Louise Longenecker llongen at unix.cc.emory.edu
Thu Dec 1 15:46:35 EST 1994


Netters, 
	We are studying Ras activity by analysis of the state of guanine 
nucleotides present.  In short we stimulate 32P-labeled cells, and lyse 
them.  The lysate is precipitated with an anti-Ras antibody.  Following several 
washes, the GTP/GDP is eluted by heating to 95C for 5 min in 2mM EDTA, 
0.2% SDS and 2mM DTT.  The nucleotides are separated by TLC on 
PEI-cellulose plates in 0.75M KH2PO4 solvent.  Our problem is this.  The 
labeled nucleotides that we get from the immunoprecipitation are 
migrating at a different (faster) rate that our standards.  The standards 
are in the same buffer that the experimental samples are eluted into.  In 
fact they migrate differently even when the standards are mixed into the 
experimental samples.  I was hoping that someone may have had a similar 
experience since this is an established procedure (Methods in Enz. Vol. 
238), and could offer some suggestions.  The experimental spots which are 
migrating more rapidly show the expected Ras activity if in fact they 
correspond to GTP and GDP.  Thanks in advance for any suggestions.

	Bill Paxton
	Emory University
	e-mail: llongen at unix.cc.emory.edu



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