Loss of Phosphorylated Protein Signal

Symptom

Phospho-specific antibody shows no or weak signal in IP experiment, while total protein antibody works. Basal phosphorylation levels appear insufficient for detection.

1. 1 Phosphatase activity during lysis degrades phosphorylation modifications rapidly
2. 2 Absence of serine/threonine phosphatase inhibitors (sodium pyrophosphate, beta-glycerophosphate)
3. 3 Absence of tyrosine phosphatase inhibitor (sodium orthovanadate) in lysis buffer
4. 4 Low basal phosphorylation levels in unstimulated cells or tissues

1. 1 Add sodium pyrophosphate (2.5 mM final) and beta-glycerophosphate (1.0 mM final) as serine/threonine phosphatase inhibitors
2. 2 Add sodium orthovanadate (2.5 mM final) to inhibit tyrosine phosphatases in lysis buffer
3. 3 Use Phosphatase Inhibitor Cocktail #5870 or Protease/Phosphatase Inhibitor Cocktail #5872
4. 4 Treat cells with kinase activators or growth factors identified via PhosphoSitePlus before lysis

Related Video (2)

Cell Signaling Technology ★ 82

Western Blotting Protocol

"Western blotting protocol from CST directly complements the IP phospho-signal failure, showing correct lysis/handling to preserve protein modifications."

Bio-Rad Laboratories ★ 62

Setting Up and Running Mini-PROTEAN® TGX™ Precast Gels

"Gel setup and running is part of the downstream immunoprecipitation/WB workflow where weak phospho signals would be visualized."