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Western Blot (Sigma Protocol) moderate

Enzyme Inhibitor Interference

Symptom
No signal or very weak signal despite proper antibody binding
Common Causes
  1. 1 Sodium azide present in buffers inhibiting peroxidase reactions
  2. 2 Over-washing removing enzyme conjugates
Solutions
  1. 1 Do not use sodium azide in buffers when using peroxidase-conjugated antibodies
  2. 2 Shorten wash times and omit detergents from washing buffers
Related Video (3)
Cell Signaling Technology ★ 85
Western Blot Troubleshooting Guide
"Dedicated Western blot troubleshooting guide directly addresses diagnosis of signal problems and solutions, core to understanding this failure case."
Bilibili (China-Accessible Mirrors) ★ 78
Western blot full protocol: Protein extraction to chemiluminescence
"Complete workflow including chemiluminescence detection step where sodium azide interference with peroxidase reactions would manifest and be observable."
Bilibili (China-Accessible Mirrors) ★ 72
Reliable and Reproducible Western Blot Results
"Technical webinar on achieving reliable Western blot results emphasizes proper reagent handling and experimental technique to avoid common pitfalls."
Source: sigmaaldrich.com ↗
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