membrane proteins

Dr E. Buxbaum EB15 at le.ac.uk
Thu Mar 20 05:34:40 EST 1997


krautwald at ita.fhg.de (Stefan Krautwald) wrote:
>Does anybody know a protocol how I can isolate a
>membrane-associated protein without loss of biological
>activity. It´s very important to get rid of the cytoplasmic
>and nuclear proteins.

Homogenise your material, than give it a 10 min spin at 10.000 times gravity. 
This will remove cell debries and the nuclei. Then spin the supernatant 30 min 
at 100.000g to pellet the membranes. Discard the supernatant (which will contain 
the cytosolic proteins) and resuspend the pellet. Load it onto a sucrose gradient 
(5-30% is a good starting point) and do a rate zonal separation. Fraction the 
gradient and monitor the marker enzymes for the differnet membrane systems in 
the cell (plasma membrane, golgi, mitochondria, lysosomes and so on). Any 
textbook on biochemical lab technique will cover the details, as this is a 
standard method.




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