Add protease inhibitor cocktail fresh (1X) or use the following protease inhibitors such as Pepstatin A (1uM), Leupeptin (10uM) and PMSF (0.1mM) when you are about to perform cell lysis. Phosphatase inhibitors will be required to be added in the lysis buffer fresh only when phosphorylation states (activation states) are being investigated. The phosphatase inhibitors include Sodium fluoride (1mM) and Sodium orthovanadate (1mM). Perform cell lysis below 4 degree C using cold RIPA buffer.