I have extracted protein from mouse brown adipose tissue and after run a SDS-PAGE. As you can see in the gel, some of the bands have a dark background (lane: 2,3,4,6) and it seems that at the top some of the sample is accumulated. I am not sure if this is due to protein degradation or something different. Also, lane 1 and 5 do not show the same band separation that would be expected since all the samples were extracted from brown adipose tissue. Can somebody help me with this?
I can't see any real problems with this gel, other than that a small part of it is missing. The different band intensities are caused by differences in the density of protein in each band and is completely normal. Lanes 1 and 5 (excluding the marker lane) are virtually identical. There is some high molecular weight protein at the top of lanes 2, 3, 4, and 6. This could be actual high molecular weight protein, or it could be aggregated protein that wasn't adequately denatured by the sample buffer.
The accumulation of substance in the well is due to proteins that are not able to migrate; a solution would be to boil your sample on a heating block (90 degrees for 3mins?). Not sure why you don't have the same seperation of proteins if you did indeed load the same amount of the same thing... The background effect can be reduced by leaving your gel in water for a few hours.
Thanks Adam and Almutasem for the answers.
Almutasem: I do heat the samples at 100oC for 5 min. I can try a fresh sample loading buffer and see if that improves.
As Adam says, there are no problems in your SDS-PAGE Your SDS-PAGE has low background, sharp band and each line was migrated directly.
Darker band=more intensity=more protein conten
if your protein concentration in each lane is similar, the density of each band can be used as value for protein content comparison.
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