crooked bands on SDS-PAGE - (Nov/20/2006 )
I've searched for a reason for this, to no avail...
When I run SDS-PAGE, my bands come out wavy and zig-zaggy. This happens with both the sample and the markers. I typically use an 8% separating gel and 3.5% stacking gel.
How do I get nice, straight bands?
a few things off the top of my head:
use good quality acrylamide
make sure that the gels are fully polymerized
make sure that the wells are well formed and flat
make sure there are no "skins" in the wells
make sure that the stack/running gel interface is uniform and straight and flat
make sure that there are no particulates in your sample
make sure that the salt concentration in your sample is low
could you use a higher percentage in your stack (4.5%)? it will be more stable mechanically and should remain straight and flat and may even polymerize better.
In addition to the above ,Use only Tris base in ur gel buffer as if u used Tris -cl the concentration of salt in buffer will be too high and polypeptides will migrate anomalously through the gel leading to diffuse bands.
Also start ur electrophoresis with 50-60V till ur bands are lined up between the 2 gels then increase it to 120 or 150, if u go to 200v the gel will finish faster but will get bad looking bands.
U can also wash ur wells with running buffer in order not to get air buubles as it will cause small circular depression in the wells after polymerization that will lead to distortion in ur protein bands.
In addition to the above recommendations, prepare again a fresh solution of SDS.
I used some commercially available solution of SDS with a tendency to precipitate (due to low pH).
i got the same kind of bands as yours. separation and analysis was OK, but the bands were not straight.
Now I prepare SDS from the powder, keep it several months at room temperature (it doesn't precipitate) and bands are really nice.