Hi

20% SDS, 1 M tris, 2% glycerol, Urea and 0.05% mercaptoethanol

You could try the RIPA buffer supplemented with proteases inhibitors. I use this preparation for my western blotting

You do not specify whether you need cytoplasmic proteins or membrane proteins. If cytosolic you first lyse cells and then perform centirfugation to remove all subcellular components. I mean that depending of what you want the buffer may be different.

Hello Utsav,

I would suggest your standard lysis buffer you have specified for general inhibition of endogenous proteolytic machinery. However to resolve your degradation issue you can make a protein inhibitor cocktail solution (10mL deionised H20, 1 Roche cOmplete protease inhibitor tablet, 10ug/mL E64D/Pepestatin A). In my own experience this was effective for performing western blots in A549, RAW264.7 & HEK293 cell lines. The important factors in this would be what protein do you want intact (for me this was the LC3B autophagy associated protein) and what cell line you are lysing. Though my lysis solution was suitable for my cell models, I don't know how well this formula will transpose to other cell models.

Best of luck

Dear Rafael Franco,

Thank you so much for your feedback. I want both cytosolic protein mostly. But yes, I want some of the nuclear protein too.

Thanks,

Utsav

Dear Rafael Franco,

Thank you so much for your feedback. I want both cytosolic protein mostly. But yes, I want some of the nuclear protein too.

Thanks,

Utsav