General?Principles?of?Immunoprecipitation

Lysis buffer.

The choice of lysis buffer depends on what the cells were labeled with and whether you want to obtain an active kinase.The lowest background is obtained with RIPA buffer without EDTA.EDTA increases the precipitation of both actin and myosin greatly.Since actin is not phosphorylated,this doesn't matter much for ³²P-labeled samples.RIPA is however more denaturing than NP40 buffer and should not be used for fragile enzymes.

³²Pi-labeled cells and isolation of protein kinases.

RIPA buffer containing phosphate buffer and supplemented with 100 to 200 µM sodium vanadate,50 mM sodium fluoride and 2 mM EDTA should be used with cells which have been labeled with ³²Pi.It is also the choice for cells from which you want to obtain proteins with protein kinase activity,provided the kinase is stable.

The EDTA should chelate all the Mg⁺⁺ in the cells and prevent phosphorylation.Phosphorylation can occur following cell lysis,with the cellular pool of ATP serving as the phosphate donor.The vanadate should inhibit most tyrosine protein phosphatases and the fluoride should inhibit some serine/threonine specific protein phosphatases.Whether the additives need to be present in all the washes isn't known.They may be completely dispensable after the first spin.

Tamara has found that the vanadate works much more effectively if it is added to the lysis buffer freshly!

Phosphate-buffered RIPA is the best choice.Phosphate is a good buffer at pH 7.2 and also functions as an inhibitor of phosphatases.