Irregularities in width of samples running on SDS-PAGE gel - (Oct/06/2010 )

I have been getting some irregularities between samples on how they are running on my SDS-PAGE gel. Specifically, I have lanes that progressively narrow as they run down the gel so that my band after Western blotting is more circular than band-like. Adjacent to these, I have lanes that do exactly the opposite; they widen as they run down the gel such that the protein band I am detecting is nearly twice the width of the lane I've loaded. These effects could be due to forced narrowing of the first set of samples or forced spreading of the second set of samples leading to compensation by the other set of wells, or a combination of both. I am not sure what could be causing my samples to run differently.

Of note, the pre-gel treatments of the two sets of samples were different. My samples that resulted in very narrow bands contain relatively concentrated protein (total cell lysate, not directly quantitated), were preciptated in TCA and washed in acetone, and were brought up in 1x SDS sample buffer diluted in dH2O. The samples that resulted in wide bands contained relatively dilute protein (one to several proteins purified from the total cell lysate used above), did not undergo the TCA precipitation and wash steps, and were brought up in 1x SDS sample buffer diluted in a buffer containing 0.5% SDS, tris, and EDTA.

A final note: the polymerization of these specific gels was mistakenly carried out using twice the recommended volume of TEMED and APS, which may have led to problems with the gels overall but I don't think would be expected to result in opposite problems between different sets of samples on the same gel.

If anyone knows of any reason the difference in my sample prep protocol could be causing the gel irregularities I would appreciate the insight; or, if there is another reason I may be overlooking please let me know! Thanks!

was there salt in the dilute, unprecipitated sample?

one of the causes of what you are seeing is salt in some of the samples.

try treating the samples the same way.

mdfenko on Wed Oct 6 19:12:47 2010 said:

The dilute samples were eluted from an immunoprecipitation and were therefore just analyzed in the elution buffer (SDS, EDTA, and Tris) plus 200mM sodium chloride - so yes, there was some salt in these samples alone which I failed to mention in my original post. That salt difference is one I hadn't considered, I can try changing my protocol so that salt is eliminated. Thanks for the input.

Hey,

I am running SDS-PAGE with some samples loading in Laemmli buffer containing 2% SDS and some in Laemmli buffer containing 5% SDS. I was surprised to see that my front lane for samples in 5% SDS is wide while the front lane of those in 2% SDS is narrow. Does someone have an idea to get narrow front lane with samples loading in 5% SDS?

Thanks,