Low Signal for Phosphorylated or Modified Proteins (Immunoprecipitation (CST Guide))

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IP of post-translationally modified proteins yields weak or no signal. Input lysate shows low basal levels of phosphorylated or modified target protein despite total protein presence. Common causes: 1) Post-translationally modified proteins expressed at low basal levels in unstimulated cells or tissues 2) Phosphatase activity degrades phosphorylation during cell lysis and processing 3) Insufficient or missing phosphatase inhibitors in lysis buffer (sodium pyrophosphate, beta-glycerophosphate, sodium orthovanadate) 4) Cell treatment insufficient to induce target modification to detectable levels Solutions: 1) Include phosphatase inhibitors in lysis buffer: sodium pyrophosphate (2.5 mM final), beta-glycerophosphate (1.0 mM final), sodium orthovanadate (2.5 mM final) 2) Use Phosphatase Inhibitor Cocktail #5870 or Protease/Phosphatase Inhibitor Cocktail #5872 3) Consult PhosphoSitePlus for literature on specific modification sites and optimal induction conditions 4) Use Control Treatments by Target table to identify validated treatment, cell line, and tissue combinations that yield detectable signal 5) Include input lysate control to verify target protein expression and modification levels