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Western Blot (Sigma Protocol) minor

Black Spots on Membrane

Symptom
Random black spots or speckles appear on the western blot membrane
Common Causes
  1. 1 Blocking reagent has clumped and antibodies are binding to aggregates
  2. 2 Contaminating enzymes present in sample or blocking buffer
Solutions
  1. 1 Filter the blocking reagent before use
  2. 2 Filter the blocking buffer to remove particulates
Related Video (3)
Cell Signaling Technology ★ 92
Western Blot Troubleshooting Guide
"Dedicated Western blot troubleshooting guide that would directly address black spots and blocking/antibody issues."
Bilibili (China-Accessible Mirrors) ★ 72
Western blot hands-on: Protein extraction through detection
"Hands-on protocol with explicit troubleshooting of common WB pitfalls, likely covering blocking and reagent handling."
Cell Signaling Technology ★ 65
Western Blotting Protocol
"Step-by-step reproducible WB protocol useful for seeing correct blocking and reagent preparation to avoid aggregate artifacts."
Source: sigmaaldrich.com ↗
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