SDS-PAGE: Protein bands broaden as it migrates down the gel like a pyramid!
Posted 18 June 2010 - 08:27 AM
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...
Posted 23 June 2010 - 08:27 AM
your picture doesn't look that bad (one or two lanes look like they have been influenced by buffer components).
genius does what it must
i do what i get paid to do
Posted 24 June 2010 - 06:48 AM
I will try to prepare fresh loading buffer, electrophoresis buffer and transfer buffer for my next trial!