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Lysis buffer for leptin receptor and pSTAT detection - (Nov/25/2013 )



I want to detect leptin receptor and pSTAT in tissue samples using western blot and would like some suggestions on lysis buffer and protein preparation procedure. I tried a buffer (20 mM Tris, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Na4P2O7 with phosphatase and protease inhibitors) generally used in the lab for WB, but could only see a faint band at the estimated size of leptin receptor. In many papers I looked at where people have detected leptin receptor on WB they used RIPA buffer. Would RIPA buffer be OK for pSTAT detection? I am considering making first a preparation of cytosolic proteins and then one for membrane proteins. What buffers are best in that case? 



Maria B

-Maria Boije-

RIPA is a general lysis buffer that is fine for most proteins.  I don't know specifically for pStat though.