dye front running funny - western blot (May/20/2006 )
Hello all,
I have been running 10-12% polyacrylamide gels and so far everytime I run the gel, my dye front is either not compressed tightly or becomes distorted. My ladder runs very good and my protein (after blotting) looks quite straight, except bands under 20KD (and only bands under 20KD) sometimes are distorted as well (protein ladder 11 and 17KD still runs perfectly well). I prepare my samples by directly adding the loading buffer to my samples, I boil them for 5min, and add to wells. Does anybody else have this problem?
Any advice is greatly appreciated.
Alan
I have been running 10-12% polyacrylamide gels and so far everytime I run the gel, my dye front is either not compressed tightly or becomes distorted. My ladder runs very good and my protein (after blotting) looks quite straight, except bands under 20KD (and only bands under 20KD) sometimes are distorted as well (protein ladder 11 and 17KD still runs perfectly well). I prepare my samples by directly adding the loading buffer to my samples, I boil them for 5min, and add to wells. Does anybody else have this problem?
Any advice is greatly appreciated.
Alan
once we had a serious problem with the running buffer (our stock had not enough SDS and pH was off). then the front dye run as a broad smeary band, and only high molecular weight protein bands on coomasie looked fine, when the low ones were totally not sharp and ugly..
I will definitely take another look at the buffer. thank you for the advice
I had something strange also : flat bands, but leaking on the side.
I prepared buffer with new SDS. Now it's perfect.