I would definitely add protease inhibitors. I would only add phosphatase inhibitors if I was trying to measure a phosphorylated antigen.

In general, you should add a protease inhibitor, while using a phosphatase inhibitor depends on your target proteins(if it is a phosphorylated protein such as Phospho-AKTfor example, you have to use a phosphatase inhibitor to preserve the phosphorylation state of proteins during and after lysis).

Hello Gokhan Gokhaner

It is necessary because proteases are present in the lysate in varying amounts and the protein of interest is susceptibility to proteolytic cleavage. Phosphatase inhibitors may be optional unless the antibodies specifically recognize phosphorylated forms of the protein.

The activity of proteases is reduced at 4°C, and it has been found in general that extraction of protein should be carried out on ice to obtain active proteins. Nevertheless, protease inhibitor cocktail should be added to the lysis buffer prior to homogenization.

for proteolytic cleavage, it is necessary to add proteinase inhibitors.

Thank you for all your contributions, they are all appreciated. In relation with that, some scientists say some kits may not be compatible with protease inhibitor cocktails. How I could avoid making any mistake when deciding whether I use it or not? Are there any specific rules or limitations regarding protease inhibitor cocktails?