wobbly bands in WB - (Oct/20/2008 )
I get wobbly bands in my western blots. I equilibriate the gel for 45 min in buffer and do all the necessary wash steps.
Can somebody please explain why is it happening and the solution for it?
Do you use precast gels or do you pour your own?
What sort of wobbly? Smiles or frowns? Individual bands wobbly?
Smiles and frowns are caused by the gel running too hot or not having enough electrolyte respectively. Individual bands can be cause by the bottom of the well being uneven, or irregularities in the gel. I wouldn't equilibrate for more than a few minutes in the WB buffer, so as to minimise diffusion of the bands, which could also result in some funny looking westerns.
I also got the same problem with the bands. I find that whenever I use the fresh transfer buffer, the bands are very nice. However, the reused buffer sometimes will give this bad result.