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.