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Western Blot (Misshapen / Uneven Bands) severe

Uneven Bands Due to Improper Gel Polymerization

Symptom
Bands appear distorted, wonky, or uneven across lanes. In extreme cases, the same protein appears at different molecular weights in different lanes due to irregular gel matrix formation.
Common Causes
  1. 1 Incorrect TEMED concentration in gel recipe causing incomplete or uneven polymerization
  2. 2 Gel not fully covered with buffer during setting process
  3. 3 Uneven polymerization across gel thickness or width
  4. 4 Temperature fluctuations during gel polymerization phase
Solutions
  1. 1 Verify gel recipe and confirm correct TEMED volume is added
  2. 2 Ensure gel is completely covered with buffer during polymerization
  3. 3 Allow adequate polymerization time (minimum 30-45 minutes for resolving gel)
  4. 4 Prepare fresh acrylamide solutions and check ammonium persulfate (APS) activity
Related Video (3)
Bio-Rad Laboratories ★ 85
Setting Up and Running Mini-PROTEAN® TGX™ Precast Gels
"Demonstrates proper setup and running of acrylamide gels with emphasis on correct procedure to ensure proper gel polymerization, directly addressing the TEMED concentration and polymerization issues."
Bio-Rad Laboratories ★ 78
Western Blotting
"Shows SDS-PAGE protein separation using Bio-Rad kit, providing context for Western blot gel preparation and the importance of proper gel matrix formation."
Bilibili (China-Accessible Mirrors) ★ 72
Western blot full protocol: Protein extraction to chemiluminescence
"Complete hands-on Western blot protocol with emphasis on troubleshooting common pitfalls, likely includes discussion of gel preparation and polymerization quality control."
Source: abcam.com ↗
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