I need a protocol for isolating cytoplasmic content from cell membrane for cell culture cell line.
Please read : http://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314807262343/litdoc28988741_20131027212557.pdf
Please read : http://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314807262343/litdoc28988741_20131027212557.pdf
If your project would allow, you could recombinantly express more of the protein of interest; would allow for better quantification.
Detergents may also be used for protein extraction; this would depend on the protein you are trying to isolate.
Dont use detergents for cell membrane disruption. It would denature proteins. Use sonication to disrupt cell membrane. Then use ammonium sulfate salting out method to get proteins
Don't use detergent! Resuspend your cells in hypotonic buffer (e.g. 10 mM Tris) and incubate on ice for 10 min. Then use 30 strokes with the "tight" pestle of a Dounce Homogenizer to disrupt the cells and centrifuge at 800g for 10 min and 4 degrees to pellet any cell debris. Take the supernatant (= cytosol + endomembranes) from this spin and ultracentrifuge it at 100.000g for 45 min and 4 degrees to pellet endomembranes. The supernatant from this spin is your cytosol.
In case your protein of interest is sticking peripherilly to membranes you can do a high salt wash of the membrane pellet, to wash off bound material and combine this with your cytosolic fraction.
Good luck!
Thank you very much Dr. Leonhardt
Is it possible to use Sonication instead of Homogenizer?
I have never tried sonication in this protocol. However, one advantage of using the Dounce Homogenizer is that this approach should leave organellar membranes intact. Hence, there should be no leakage of e.g. lumenal, soluble ER proteins into your cytosolic fraction. With sonication, I am wondering whether such leakage would occur ...
There are sequential protein extraction kits available. For example, Bio-Rad laboratories has a sequential protein extraction kit that allows for the specific extraction of the cytoplasmic proteins and the membrane proteins. The proteins thus extracted are in a form that are ready for analysis by SDS PAGE and also 2D gel electrophoresis.
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