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Homogenous Cell Lysates

Western Blot Cell Lysates Membrane proteins Loading

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#1 asta6801

asta6801

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Posted 01 April 2014 - 03:58 PM

Hi there

 

I'm having issues with some of my lysates. I get huge variation in my protein estimations and therefore my beta actin when I run the gel. I don't believe it is my pipetting or loading, as I don't have this issue with other lysates and my standard curves for the protein estimations are pretty much perfect. I think that the issue is because the lysates are not homogenous or they are too sticky/thick. I am extracting membrane proteins (specifically Pgp, MRP1 and ABCG2) with the following protocol:

 

Add lysis buffer (RIPA buffer + protease inhibitor and 0.1% SDS) to plated cells and place on rocker at 4 degrees C for 30 mins. Sonicate, then spin at 13.2 RPM for 15 mins. Store supernatant at -80 until ready to use. 

 

Does anyone have any suggestions to improve this problem? Or even another extraction protocol that they have had success with for these proteins?



#2 Curtis

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Posted 02 April 2014 - 09:12 AM

if i get sticky lysate i add DNase.







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