SDS-PAGE gel trouble shooting : Collins Lab Blog Life in the Collins lab

SDS-PAGE gel trouble shooting

One of the favorite quotes in the lab is “the gel never lies”. This is a handy compiled list adapted from “gradigel” product literature.

Problem: Distorted protein bands.

Cause: Air bubbles in the sample wells, or between gel and cassette, or at the bottom of the cassette.

Solution: Use a transfer pipette to displace the air bubbles from the sample wells.

Problem: Streaking.

Cause: Poorly soluble or weakly charged particles (such as carbohydrates) in sample.

Solution:
1. Centrifuge sample.

2. Change pH of buffer.

3. Heat sample in the presence of SDS.

Problem: Bands difficult to distinguish.

Cause: Incorrect gel selection, sample overloading, insufficient cooling buffer.

Solution:

1. Reduce sample size.

2. Select a gel which separates in the desired molecular weight range.

3. For proteins of similar molecular weight a longer gel may be useful.

4. Increase buffer in outer tank.

Problem: Sample spreading across the gel.

Cause: Too much salt in the sample.

Solution: Reduce salt by dialysis or ultra-filtration.

Problem: Sample contains appreciable carbohydrate.

Solution: Enzymatically remove the carbohydrate.

Problem: Sample contains lipoproteins.

Solution: Use a gel with large pore size at top. Try addition of a non-ionic detergent.

Problem: Protein denaturation and band inversion.

Cause: Excessive heating.

Solution: Always start with chilled buffer (<15°C).

Problem: Diffuse protein zones in gel after staining.

Cause: SDS still present in gel.

Solution: Fix in 10% TCA prior to staining, otherwise use 30% methanol in the destaining solution.

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