Bands Appear Lower Than Expected Molecular Weight

Symptom

All protein bands or the target band migrate further down the membrane than expected based on the predicted molecular weight. Coomassie blue staining of the membrane confirms abnormally low band positions before immunostaining.

1. 1 Gel electrophoresis run time exceeded manufacturer recommendations, allowing proteins to over-migrate through the gel matrix
2. 2 Insufficient acrylamide percentage in the gel for the target protein molecular weight, reducing matrix resistance
3. 3 Excessive voltage (>150V) applied during electrophoresis, accelerating protein migration beyond optimal separation
4. 4 Running buffer temperature too high, reducing gel viscosity and increasing migration speed

1. 1 Run gel at 100-150V for 1-2 hours in pre-chilled electrophoresis buffer according to manufacturer instructions
2. 2 Frequently monitor ladder marker separation to stop electrophoresis when bands are adequately resolved within target protein size range
3. 3 Use higher acrylamide percentage gels (12-15%) for lower molecular weight proteins (<50 kDa)
4. 4 Verify Coomassie blue staining of transferred membrane before proceeding with blocking to confirm proper band positions

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