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Study Notes: SDS-PAGE Troubleshooting

The technique of SDS-PAGE seems simplistic with so few pieces of equipment interacting to analyse a sample and to produce a gel as a result. But looks can be deceiving! The emphasis on hands-on preparation of gel solutions, pouring of the gels, and the assembly and running of the samples has the potential to lead to a number of common and not so common problems. In such a system the potential for things to go wrong is necessarily high if the operator does not know what they are doing or is less than careful in their approach to their work.

But even for experienced and meticulous technicians things can and do go wrong! This is where the ability to ‘troubleshoot’ is important.

The following table contains examples of errors/malfunctions and the likely appearance of the equipment or the resultant gel under these circumstances. These examples will be useful when you work with the virtual SDS-PAGE.

Note that this is not an exhaustive list and there may be other errors/malfunctions that may occur.

Effect on apparatus/gel
Assembling gel sandwich incorrectly Cracked or leaking plates
Leaking of acrylamide from the gel plates prior to polymerisation Distortion of bands on the gel
Leaving TEMED and Ammonium Persulfate out of the gel mixture Gel will not polymerise.
Incorrect buffers in stacking/separating gels Migration of bands impeded, distorted bands
Incorrect running buffer in tanks Migration of bands impeded, distorted bands
No buffer in tanks No current - gel will not run
Powerpack not turned on No current - gel will not run
Leads around the wrong way Sample migrates out the top of the sample wells.
Current too high Overheating, ‘smiling’ of gels
Too much sample Smearing of bands
Sample added to well without care Mixing of sample with adjacent well and mixing of sample
SDS not added to sample No net negative charge on proteins, aberrant migration of bands
DTT not added to sample Protein complexes not disrupted - unusual high Mr bands appear in gel
Sample not added to well No bands in the gel
Staining of gel not done correctly Poorly stained or no bands apparent in gel


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