SDS-PAGE: Protein bands broaden as it migrates down the gel like a pyramid! - (Jun/18/2010 )

Dear all,

I am wondering if any of you had similar experience. Occasionally, i find my protein bands at the edge of the gel to broaden as it migrates down the gel. The small protein hence have a very WIDE band whereas the heavy protein has a thinner band. The protein sample in the middle of the gel remained rather constant though. It always happen only to the 2 lanes at both ends of the gel. In theory, i think that's not unexpected as the pressure exerted by the protein from the middle will force the ones at the edge towards the side. I won't mind if it's less obvious but mine is SUPER obvious!

My running conditions to detect a protein at the size of 50 kDa are:
1. 5% stacking gel + 10% resolving gel.
2. 100 V constant till dye front almost come to the bottom; running buffer is cold.
3. Protein lysed in 1x SDS buffer, loaded 20 ul.

I asked around and some seemed to suggest that i have loaded too much protein hence the pressure is very high against those at the edge. But i have no choice as my protein of interest is not that abundant in my cells...

this effect is seen often. it is caused by factors such as uneven heating of the gel (center warmer than the sides, adds that nice smile to the gel), unchecked diffusion of proteins in end lanes (path of least resistance), salts and other buffer components in the sample,...

your picture doesn't look that bad (one or two lanes look like they have been influenced by buffer components).

Thanks for your reply mdfenko!

I will try to prepare fresh loading buffer, electrophoresis buffer and transfer buffer for my next trial!