I am having problems with some samples in SDS-PAGE. For 2 specific proteins (one viral protein one serum protein) I am getting almost consistent unexpected bands across the gel. I have included the picture. The first 6 protein lanes are 6 different constructs of the same protein on different carriers (non-reducing) the next 6 after the ladder are the same but reducing. The last two wells are NR & R of a different protein. Each was purified by a different immunoaffinity column, concentrated in a different concentrator, etc... I also run MANY other proteins day to day that do not have these bands. For some reason it is only these two unrelated proteins. I have considered uneven cooling, but that doesnt seem to make sense for this scenario. The 12 lanes should show a band around 50 and around 15 (the darkest clean bands) and the last 2 should show up at those darkest bands as well. I'm just concerned about the faint bands.
I ran it at 150V for 1 hour with slightly cooled buffer (last minute run).
Any advice/thoughts?
To me the gel looks fairly normal for a protein from an affinity column: a major band from the target protein and several weaker ones from proteins that either also bind to the affinity matrix, or that form a complex with the target protein. You need to follow with some polishing steps, for example IEC or HIC followed by SEC.
Other than that, making the stacking gel a little longer (5 mm or so) may give you better focusing of bands.
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