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]

Failed Gels - Crooked, Broken, Smeared, Weird

Crooked bands

Examples of this problem are scattered throughout the 'hall of shame.' The surface of the resolving gel was uneven, so that when the samples were stacked, the bands started out with distorted shapes.

Crooked bands also result from an uneven electric field, but usually that type of distortion is symmetrical.

Bad wells

If samples run over into adjacent wells, you will see what looks like a continuous protein band running across the wells (arrow). In fact, that's exactly what it is. This is usually what happens when the wells are too short, or perhaps sample is accidentally spilled outside the wells.

In addition, we may have used old sample buffer. An advantage of using Cleland's reagent (dithithreitol, or DTT) to reduce disulfide bonds is that it doesn't stink as badly as 2-mercaptoethanol. However, the latter compound is much more stable. Disulfide bonds link individual polypeptides together and maintain some proteins in a folded state under otherwise denaturing conditions. This causes them to run unpredictably.


Dense Gel

...or should it be dense investigators? The gel mix was clearly incorrect. The acrylamide concentration was so high that only the smallest proteins entered the gel to any extent. The bands were distorted due to overloading and the fact that beyond a reasonable concentration the most concentrated gels tend not to polymerize evenly.


Broken Gel

This can be really frustrating. You did a very nice job, then the gel broke during handling. Keep in mind that the gel will still be usable as long as you save the pieces.

Field Effects

To some extent this is normal, although for some reason the field effect was exaggerated here. The spacers interrupt the electric field at the edges of the gel, so that the 'pull' (or 'push' if you prefer) is biased to one side the closer the sample is to the edge of the gel. The result is the 'frowning'effect you see here.

The individual bands sometimes smile, but if the gels have any expression at all, they always frown. I guess you would too if we zapped you with electricity and drowned you in blue dye.



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Created by David R. Caprette (caprette@rice.edu), Rice University 9 Oct 96
Updated 4 Dec 06