plasma membrane extraction

Dr Engelbert Buxbaum engelbert_buxbaum at hotmail.com
Wed Feb 23 07:07:56 EST 2005


Tien T Nguyen wrote:

> Hello,
> 
> I have been having problems with extracting plasma membrane from the
> aorta, for western blotting; unlike the heart, I was unable to detect my
> receptor from the aorta. Any suggestions?
> 
> I used the following method to extract the membrane from both blood vessel and heart. 
> 
> The tissues were crushed to powder and transferred to ice-cold buffer
> (50mM HEPES, 150mM NaCl, 1.5mM MgCl2, 10% glycerol, 1% triton X-100 and
> 20mM PMSF) for 30min on ice and homogenised in a Polytron homogeniser
> (at 4oC). The homogenate was centrifuged at 16 000 rpm for 15min at 4oC,
> and the supernatant was collected.

I am afraid this is not enough info for us to help you. Did the
supernatant contain any protein? Were protein bands detectable on your
blot with a general protein stain (CBB, Ponceau, colloidal gold)? What
receptor did you look for in your blots, and is it supposed to be
expressed in aorta? Did you blot for housekeeping proteins (actin,
Na/K-ATPase or similar)? You say you crushed the tissue: was it
lyophilised or frozen? The inclusion of SDS in the extraction buffer may
increase its stripping power. 

Just a couple of thoughts about potential problems. Eliminate them
systematically.



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