Hi everybody,
I would like to check wheather my protein is located in plasma membrane. Can anyone recommend an efficient procedure for isolation of plasma membrane proteins from animal cells cultured in vitro? The protein of interest will be identified using Western blotting method. Thank you for any help you can provide for this question.
Sucrose density gradient ultracentrifugation is a way to separate the various organelle membranes.
We used a ball bearing homogenizer in 0.25 M sucrose, 5mM MgCl2, 25mM Tris, pH8, protease inhibitors, to break open the cells, or you can freeze thaw to break them. If it is animal tissue, use a dounce homogenizer. Spin at at 100,000 xg. For tissue, spin at 2000 xg and discard pellet, and then do the 100,000 xg spin.Take the pellet,from the 100,000 xg spin and Resuspend in the same buffer without magnesium. Prepare a sucrose gradient in a centrifuge tube. 28%-45% w/w. Use a swinging bucket rotor, and centrifuge o/n in the cold. Collect membranes from each interface, dilute out with buffer to dilute the sucrose, spin at 100000 xg to pellet. Resuspend the pellets in the 0.25M sucrose, then assay each fraction for plasma membrane markers. The PM fraction is usually at the top of the gradient.
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