I hope somebody will be able to help us with this. In our lab we are experiencing difficulties with our western blots, and we think there is a problem with our Loading buffer. The thing is that we are obtaining separated bands where we should see only one band, for example with tubulin. We think there is a problem with the compactation of the sample. We have tested different buffers (I'll write the recipies at the bottom), and the first days that we use them there's no problem: we have defined bands and everything is OK. But two or three weeks after we start having the same problem.
The first buffer we tested was:
Tris pH 8 0.03 M
Glycerol 30 %
SDS 7.5 %
DTT (Dithiothreitol) 0.15 %
Bromphenol blue 0.05 %
Then we tried with the one that is given in this page (Molecular Station):
Sample Buffer (4X Laemmli Buffer)
* 2.4 ml 1 M Tris pH 6.8 (Same as upper gel buffer)
* 0.8 g SDS stock
* 4 ml 100% glycerol
* 0.01% bromophenol blue. Final Concentration is .02%
* 1 ml ß-mercaptoethanol (electrophoresis grade)
* 2.8 ml water
And as I said the first days everything went right, but then we get horrible western bands.
I guess maybe we are doing something wrong when we prepare this buffers, or maybe we store them in the wrong conditions.. We have tried storing them at -20C and at RT, and the problem occurs in the two cases.
Could someone give us some advice on how to prepare and store this buffers? Do we have to heat them a bit before using them? Is it normal to have foam due to the SDS? Or maybe someone thinks the problem is not on the buffer but on another thing...
Any comment will be welcome. Thanks in advance and I'm sorry if something it's not clear or English is not correct.