Home › Failure Case Library › Enzyme Inhibitor Interference
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 ★ 92
Western Blot Troubleshooting Guide
"Direct troubleshooting guide addressing signal problems and failure diagnosis in Western blotting, most likely to cover buffer component issues including sodium azide interference."
Bilibili (China-Accessible Mirrors) ★ 78
Western blot full protocol: Protein extraction to chemiluminescence
"Complete hands-on workflow including chemiluminescence detection step where peroxidase inhibition manifests as weak/no signal, provides context for identifying reagent-related failures."
Bilibili (China-Accessible Mirrors) ★ 72
Reliable and Reproducible Western Blot Results
"CST technical webinar on reliable and reproducible results emphasizes proper experimental technique and likely covers buffer preparation and reagent quality control to avoid inhibition artifacts."
Source: sigmaaldrich.com ↗
← Back to all cases