I want to separate the plasma membrane fraction from the cultured muscle cells upon sample treatment in order to analyze Glut4 translocation into the plasma membrane. I usually extract the cells with 0.1% NP-40 buffer followed by a homogenization step using a handheld pestle homogenizer. The homogenization is carried out for 30 s followed by 10s interval, which is repeated 5 times. Then the cell homogenate is centrifuged and the supernatant is collected as the cytosolic protein fraction. The precipitate is retreated with 1% NP-40 buffer after incubating on ice for 1h. Then the plasma membrane fraction is collected as the supernatant. Glut4 translocation is determined by western blotting. However, I often find the reference protein for plasma membrane (IR-beta) in the cytosolic protein fraction. Is this due to the homogenization step? If so, what is the optimal conditions for homogenization? Prolong or just one or two strokes? Or else, how could I optimize the protocol to ensure a complete extraction of plasma membrane proteins?
Try this. Break the cells by sonication in a buffer that has no detergent. Ultracentrifuge the broken cells. Homogenize the pellet in a buffer that also has no detergent. Apply the homogenized pellet to a sucrose step gradient with a 16%/31% sucrose interface. Ultracentrifuge overnight in a swinging bucket rotor. Collect the purified plasma membranes from the 16%/31% sucrose interface. Extract that material with whatever method suits your needs. You may need a stronger detergent than NP-40. If you are just going to run an SDS-PAGE gel, use SDS. If you need native protein, try a zwitterionic detergent such as Zwittergent 3-12.
More details can be found here: http://www.jbc.org/content/269/5/3745.long
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