Loss of Phosphorylated Protein Signal (Immunoprecipitation (CST Guide))

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Phospho-specific antibody shows no or weak signal in IP experiment, while total protein antibody works. Basal phosphorylation levels appear insufficient for detection. Common causes: 1) Phosphatase activity during lysis degrades phosphorylation modifications rapidly 2) Absence of serine/threonine phosphatase inhibitors (sodium pyrophosphate, beta-glycerophosphate) 3) Absence of tyrosine phosphatase inhibitor (sodium orthovanadate) in lysis buffer 4) Low basal phosphorylation levels in unstimulated cells or tissues Solutions: 1) Add sodium pyrophosphate (2.5 mM final) and beta-glycerophosphate (1.0 mM final) as serine/threonine phosphatase inhibitors 2) Add sodium orthovanadate (2.5 mM final) to inhibit tyrosine phosphatases in lysis buffer 3) Use Phosphatase Inhibitor Cocktail #5870 or Protease/Phosphatase Inhibitor Cocktail #5872 4) Treat cells with kinase activators or growth factors identified via PhosphoSitePlus before lysis