smear of loading buffer observed - SDS-PAGE gel (Mar/30/2005 )
These days I found a strange thing happened during SDS-PAGE gel running. I found the blue dye is diffusing, forming big smear. At the same time, the pre-stained protein marker was sperated well in the resolving gel, because I can clearly see the ladders. After SDS-PAGE gel running, when I finished western blot, I can see my protein of interest as a single band, with expected size. So it seem that this blue dye diffusion does not affect SDS-PAGE.
But this phenomenon only appears recently, before this, the blue dye forms a very nice front line during gel running, it is very thin, crossing the whole gel.
I have changed everything such as loading buffer, running buffer, also, thoes reagent that used in preparing gel. But I still observed the same thing.
Did anyone experience this before? Although it doesn't affect the result, I'm very curious to know the reason.
Thank you very much:)
i just get this probmem yesterday. I suppose mine came of a too low laemmli concentration in the sample i loaded...
I think this may be due to the Change in pH in the stacking gel, thus lower the stacking power, so the dye front smear.
You said that you were not affected by this. I guess that because you are not dealing with a small protein and i think small protein need more stacking power than large proteins in order to be stacked to a nice band.
I am working with a 7kDa protein, if i saw the dye front form smear, my protein usually did not stack well.
Hi may I know how to increase the stacking of protein, I have 12kD protein and it does form smear below.
You can begin reducing the acrylamide concentration of your running gel by 1%. That would tend to separate the SDS-small proteins complexes from the SDS micelles.
you can try switching buffer systems from tris-glycine to tris-tricine. this is great for smaller proteins and peptides.