IgG Heavy/Light Chains Obscure Target Signal

Symptom

Strong bands at approximately 50 kDa (heavy chain) and 25 kDa (light chain) obscure target protein signal. Same host species used for IP antibody and western blot detection antibody.

1. 1 HRP-conjugated secondary antibody detects both denatured IgG chains from IP and native primary antibody
2. 2 Same species antibodies used for IP pull-down and western blot detection create cross-reactivity
3. 3 Denatured IgG heavy chain (50 kDa) and light chain (25 kDa) run at high abundance masking nearby target bands
4. 4 Non-specific secondary antibody binds all IgG regardless of native or denatured state

1. 1 Use different species antibodies for IP and western: rabbit for IP with mouse for western (or vice versa) with species-specific secondaries (Anti-rabbit IgG HRP #7074, Anti-mouse IgG HRP #7076)
2. 2 Use biotinylated primary antibody for western detection with Streptavidin-HRP #3999 (no IgG cross-reactivity)
3. 3 Use light-chain specific secondary: Mouse Anti-Rabbit IgG Light-Chain Specific (D4W3E) HRP #93702 or Rabbit Anti-Mouse IgG Light-Chain Specific (D3V2A) HRP #58802 (only if target >30 kDa)
4. 4 Use conformation-specific detection: Mouse Anti-Rabbit IgG Conformation Specific (L27A9) mAb #5127 or Protein A HRP Conjugate #12291 (preferentially binds native IgG, minimal denatured IgG reactivity at low concentrations)

Related Video (2)

Cell Signaling Technology ★ 85

Western Blotting Protocol

"Directly shows the western blot workflow, including antibody incubation and detection steps where the IgG chain interference occurs."

Bio-Rad Laboratories ★ 60

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

"Demonstrates gel electrophoresis and protein separation, helping visualize where heavy and light chains run relative to target bands."