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Homogenize the cells or tissue of interest in lysis buffer made fresh and containing a cocktail of protease inhibitors (and phosphatase inhibitors when dealing with phosphorylated proteins).
As soon as lysis occurs, proteolysis, dephosphorylation and denaturation begin. These events can be slowed down tremendously if samples are kept on ice or at 4°C at all times and appropriate inhibitors are added fresh to the lysis buffer.
Use a RIPA or NP40 buffer supplemented with fresh protease and phosphatase inhibitor cocktails.
Remember to add phosphatase inhibitors to cocktails bought when investigating phosphorylation events.
Keep the proteins in their phosphorylated state! Add adequate phosphatase inhibitors and keep samples on ice at all times.
Block the membrane in 5% w/v BSA (fractionV) NOT MILK (milk contains casein which is a phosphoprotein; this results in high background as the phospho-specific antibody detects the casein present in the milk).
Remember the phosphorylation may need to be induced. Low signal or no signal may mean that the induction is not sufficient. Run the recommended positive control with your samples.