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]

Smeared Protein Gels

Smeared gels – example 1

Smearing can have a variety of causes, but most commonly it is due to an unevenly poured acrylamide mixture or due to gross overloading of protein.

In this example the gel was not properly poured, so that the lower half had begun to polymerize before the upper part was poured. The first gel mix began to polymerize too quickly. Rather than prepare a new gel cassette, the students simply stopped pouring, prepared a new mix, and poured it on top of the old. Obviously, the bond wasn't particularly good.

Smeared gels – example 2

In this example a lot of protein was loaded in each of the wells, all the way across. Most of the lanes can still be interpreted, but smearing is particularly evident in lanes 3 and 4. Those lanes contained primarily one protein (hemoglobin subunit), as did lanes 9 and 10. However, the group that loaded 3 and 4 underestimated the protein concentrations in their red cell lysate and red cell cytoplasm fractions. Those fractions contained so much protein that samples had to be diluted prior to preparing a protein assay tube. The students forgot to take the dilution factor into consideration when determining protein concentration.

The capacity of a mini-gel for a mixed protein sample is 20 to 40 micrograms/well, depending on the resolution needed and number individual polypeptides in the mix. However, if a pure protein is loaded, one single band will contain all 20-40 micrograms, and the result is a mess.

Smeared gels – example 3

Here is another example of gross overloading, and it appears these students made the same mistake as in example 2. It looks as though there was some inconsistency in the gel also.

Smeared gels – example 4

Smearing can be a normal consequence of running membrane-associated proteins with a high lipid content in a discontinuous gel. In discontinuous PAGE, sample proteins are super-concentrated by a combination of two factors. First, the rate of migration is suddenly slowed as the sample moves from a non-restrictive stacking gel to a separating gel that restricts movement. Second (and more important), a pH change affects the electrophoretic mobility of proteins in the sample, causing the sample to check up abruptly. A sample of a half cm or so in height becomes compressed into a layer of bands a few micrometers thick, and the local concentration of protein goes up considerably.

Membrane-associated proteins tend to precipate at lower concentrations, thus lanes containing such proteins often have a dark band of precipitated proteins at the very top. As electrophoresis proceeds the precipates re-dissolve and enter the gel continuously, thus forming a continuous dark background of unresolved polypeptides. Many of the membrane samples shown on these gels will show that characteristic.

The resolved bands in the membrane protein lanes (4 and 7) appear to consist of similar amounts of protein, however in lane 4 the capacity of the gel for at least some of the membrane proteins was exceeded, resulting in a dark smear.



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