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Protocol: Isolation of Membrane Proteins

Preparation of Plasma Membranes

The following method has been optimized for use with mammalian tissues.  Other sample types may require a more severe method for initial breakage of cells.

• Mince tissue into small pieces using a very clean scalpel.  Transfer the pieces to a Potter-Elvehjem homogenizer on ice. 
• Add 2 ml cold homogenization buffer (250 mM sucrose, 1 mM EDTA, 10 mM TrisHCl buffer, pH 7.2 plus protease inhibitors) to the homogenizer.  Homogenize the tissue using approximately 20 manual up and down strokes. 
• Remove intact cells and cell debris by centrifugation at 500 x g for 10 minutes at 4˚C.  (This step may be repeated if necessary.) 
• Centrifuge the supernatant at 15,000 x g for 20 minutes at 4˚C to remove mitochondria.  Wash the resulting pellet with cold homogenization buffer and spin as before. The pellet from the last centrifugation may be saved as the “mitochondria” pellet. 
• Centrifuge the 15,000 x g supernatant from the first 15,000 x g spin for 1 hour at 4˚C at 100,000 x g to pellet the plasma membranes.  Wash the pellet with homogenization buffer and centrifuge at 100,000 x g, 4˚C, for 1 hour.  The final pellet is saved as the “membrane pellet."
• Proteins may be extracted from the membrane pellet using a rehydration buffer with strong detergents, such as Bio-Rad’s Sequential Extraction Reagent 3 or a solution of 7 M urea, 2 M thiourea, 2% ASB-14 (amidosulfobetaine 14).  Allow the pellet to incubate with the buffer for at least one hour with occasional vortexing and then centrifuge at 100,000 x g, 4˚C, for 1 hour.  Remove and save the supernatant.