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Membrane vs. Cytosol Fractionation


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#1 tabassum.sharma

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Posted 03 April 2013 - 12:27 PM

Hello,

I am trying to perform fractionations on brain tissue to extract membrane and cytosolic proteins, however the biomarker that we used as a control, GAPDH, is showing up in the membrane when we perform Westerns, which we believe to mean that the membrane fraction is contaminated with the cytosol. Is there another biomarker that can be used as a control so that we can verify if there really is a contamination? Would anyone have any suggestions for fractionation protocols that completely isolate the membrane proteins from the cytosol and include recipes for buffers that are used and the spin speeds and time for centrifugation?

Thanks

#2 Curtis

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Posted 08 May 2013 - 12:50 AM

check Afshin Samali et al papers. I think he was from Ireland.

#3 labtastic

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Posted 14 May 2013 - 09:35 AM

How many times are you washing your membranes? If you aren't washing them after isolation, then this is the source of contamination.

Also, what is in your buffer? Try using things that will minimize non-specific protein-protein interactions (e.g. high salt concentrations [0.5M NaCl]), which may be pulling down some trace amounts of GAPDH with your membranes.




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