Hi all,
I use RIPA + PI cocktail (9:1) to lyse infected/uninfected mammalian cells (macrophages and BMDCs mostly), followed by a mechanical shearing of the cell membrane (by scratching the tube on a rough surface) for a crude cell lysate preparation.
Will this take out the nuclear proteins as well or nuclear fractionation (i dont want separate cytosolic and nuclear fraction) requires even harsh method?
RIPA buffer is what you to use if you want to solubilize all membranes. Lyse your cells with buffer and you will release all proteins within compartments, including nuclear and mitochondria proteins. This is due to the combination of harsh denaturing , ionic detergents ( sodium deoxycholate and SDS ) and the milder, non ionic detergent ( NP - 40 ) For more information consult https://advansta.com
Yes, with RIPA buffer you are disupting all membranes inluding nucleus. Thus, after mechanical homogeneization, in your RIPA buffer you will have all cellular proteins released. After that, you can remove debris by doing centrifuge
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