Overview: [Characterization of red cell membrane proteins by SDS-PAGE] [research paper]
Topics: [gel analysis] [molecular mass standard curve] [measuring relative mobility] ["Hall of Shame"]
Data: [gel images]

Bands Too Light

Light bands – example 1

It looks like this problem was caused by badly formed wells and/or careless loading of sample onto the gel. The background is dark and uneven, suggesting that protein was in the running buffer itself. Most likely the apparatus was jarred and much of the samples was slopped out of the wells. The spilled sample would not resolve into bands, since it continuously entered the gel from the upper buffer compartment.

Note the presence of a horizontal line, continuous along several lanes (arrow). That would suggest that the wells were too shallow, and some material spilled into adjacent wells.

This symptom also shows up in perfectly good gels when the electrodes are hooked up backwards for a few minutes. Protein migrates out of the wells instead of into the stacking gel. Catching the problem within a few minutes salvages some of the information, but the pH effect that causes efficient stacking is compromised, sample is lost, and protein contaminates the running buffer.

Light bands – example 2

Judging from the lack of other symptoms, it appears that the gel simply didn't stain well. Coomassie blue dye staining solution can become contaminated with SDS if it is recycled. The dye becomes less effective and proteins don't show up as well as with fresh dye. As long as the proteins were precipitated in the gel by the acidified alcohol in the stain solution, the problem could be corrected simply by re-staining with fresh dye.

Light bands – example 3

The 'X' marks the lane next to lane 5 where the amount of protein was overestimated in the original sample, the sample was prepared to the wrong concentration for electrophoresis, or too little was loaded - perhaps sample wasn't properly drawn up into the syringe. The absence of any major bands, compared to the other samples, suggests that the well was simply underloaded.

Light bands – example 4

This is one of the more disappointing symptoms you might encounter. Let's say you were extremely meticulous, did everything perfectly, and ran your gel so that the dye front was perfectly even and right at the bottom. You rinsed the gel in deionized water, then left it on the shaker. Being late in the day, you forgot to replace the water with the stain. It was stained the next day, but here's the result:

Notice that the smaller proteins diffused rather quickly out of the gel, while the larger ones at the top diffused much more slowly, and took the stain. The acidified alcohol in the stain solution and in the destain solutions is essential, as it precipates proteins, preventing them from diffusing out of the acrylamide matrix.



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Created by David R. Caprette (caprette@rice.edu), Rice University 9 Oct 96
Updated 26 May 05