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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 ★ 88
Western Blot Troubleshooting Guide
"Dedicated Western blot troubleshooting guide that helps diagnose weak/no-signal problems including buffer and reagent interference."
Bilibili (China-Accessible Mirrors) ★ 82
Western blot hands-on: Protein extraction through detection
"Hands-on full protocol with explicit troubleshooting of common WB pitfalls, directly relevant to diagnosing signal-loss causes like inhibitor interference."
Bilibili (China-Accessible Mirrors) ★ 72
Western blot full protocol: Protein extraction to chemiluminescence
"Complete workflow through chemiluminescence detection, giving context for where peroxidase-inhibiting buffer components affect final signal."
Source: sigmaaldrich.com ↗
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