Low/No Signal from Insufficient Protein Expression (Immunoprecipitation (CST Guide))

Question

Accepted Answer

No detectable signal in IP experiment. Input lysate control shows weak or absent target protein band on western blot. Common causes: 1) Target protein or interacting proteins expressed below western blot detection threshold 2) Incorrect cell line or tissue model with insufficient endogenous protein expression 3) Lack of appropriate cellular treatment or growth conditions to induce protein expression 4) Post-translationally modified proteins present at low basal levels without stimulation Solutions: 1) Use BioGPS or The Human Protein Atlas to verify expected protein expression in chosen cell/tissue model 2) Include positive control from Control Treatments by Target table to confirm experimental setup 3) Use PhosphoSitePlus to identify chemical modulators or treatments that increase modified protein levels 4) Include input lysate control at higher loading (e.g. 10-20 µg) to ensure target detection capability