Why Consistent Band Distortion happen in Lower Region of Western Blot Gel?

23 May 2025 1 9K Report

Over the past three months, I have consistently observed band distortion in the lower part of the gel after transfer in my Western blot experiments, as shown in the uploaded images. The gel preparation and electrophoresis steps were carried out as follows:

Preparation of 8% Running Gel (5 ml total) using 30% acrylamide Deionized water: 2.3 ml 30% Acrylamide mix: 1.3 ml 1.5 M Tris (pH 8.8): 1.3 ml 10% SDS: 0.05 ml 10% APS: 0.05 ml TEMED: 0.006 ml

Preparation of Stacking Gel (2 ml total) Deionized water: 1.4 ml 30% Acrylamide mix: 0.33 ml 1.0 M Tris (pH 6.8): 0.25 ml 10% SDS: 0.02 ml 10% APS: 0.02 ml TEMED: 0.002 ml

Reagents used:

30% acrylamide: GenDepot A0418-050
Tris: Duchefa T1501.1000
Glycine: Duchefa G0709.1000
SDS: Fisher Bioreagents BP166-500
TEMED: GenDepot T0512-002

Gel casting was performed using Bio-Rad gel casting equipment.

Electrophoresis was carried out using Bio-Rad equipment with the following running buffer composition: 10x running buffer (25 mM Tris 30 g, 192 mM glycine 144 g, SDS 10 g, total volume 1000 ml), diluted 1:10 prior to use.

Despite using the same protocol and reagents as other members of my lab, only my gels consistently show this specific band distortion, while others do not encounter this issue.

I would greatly appreciate any insights or troubleshooting suggestions regarding this problem.

Tomás Palmeyro

It's look like the electrophoresis equipment burn or smash the agar.. try with another electrophoresis equipment and check the results, i thought is damage back the cube maybe oxide. have a nice day.

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