Hello Anna Ferenc

RIPA lysis buffer is used for rapid and efficient total cell lysis and solubilization of proteins from both adherent and suspension cultured mammalian cells, effectively extracting cytoplasmic, nuclear and membrane proteins.

Did you add protease inhibitor cocktail to the RIPA buffer fresh at the time of cell lysis? As soon as lysis occurs, proteolysis, dephosphorylation and denaturation begin. These events can be slowed down significantly if samples are kept on ice or at 4°C at all times, and appropriate inhibitors are added fresh to the lysis buffer.

Protease Inhibitor Cocktail protects protein extracts from aminopeptidases, metalloproteases, and serine, cysteine, and aspartic acid proteases, thereby preventing proteolytic degradation during cell lysis and protein extraction. If you are interested in phosphorylated protein, you should also add phosphatase inhibitors like sodium fluoride and sodium orthovanadate as phosphatase inhibitors preserve the phosphorylation state of proteins.

Also, the number of cells used to extract protein is important.

I have used RIPA buffer for lysis of mammalian cells without any problems.

Best.

Pietro Poggio Pietro, maybe you have some suggestions ? :)

Thank you Malcolm Nobre fro your answer. Of course, I use protease and phosphatase inhibitors and I add them to the RIPA buffer not long before cell lysis. Next time I will try with higher number of cells.

I never have problems isolating protein from animal cells with RIPA buffer but these cells are problematic.