Dumbbell-shaped bands on the WB - (Jun/20/2015 )
Hi everyone,
I am experiencing lately some issues with the shape of the bands on the WB.
I asked our technician in the lab and she told me that what I am getting is called a dumbbell-shaped band. However, he couldn't come up with an explanation as to what might be causing this.
I recently realized that the problem might already be starting when I load the samples into the stacking gel. The samples do not settle very well in the gel and are somehow attracted to the sides of the well so that the sample is less in the middle and more on the sides. As a result when I start running the gel (90 Volts), the sample being on the sides tends to stay behind (although not completely) the sample in the middle .
This effect is also visible on the Ponceau staining.
If you look carefully at the attached picture there is something like a white streak running vertically through the middle of the bands and that's what I am getting on the final blot.
Does anybody have any idea why this might be happening? Could it be that the running buffer is not all the way to the top of the cassette when I load the samples?
Any advice would be greatly appreciated.
Thanks in advance!
the well forming comb may not be tight which will allow gel to polymerize between the tooth and plate. this produces "skins" which will prevent the even loading of the sample into the well.
you should clean the "skins" from the wells prior to loading. sometimes they can be flushed out sometimes they need to be scraped out. when they are gone the sample should load evenly.
one other thing can cause this effect. if you let the samples sit in the wells too long before starting the run, the sample will start to diffuse sideways. you should always start the run immediately after completing loading.
I realized the existence of these "skins" yesterday for the first time. It is really hard to see them but I think it's a good point.
I also realize that it is important to not let the samples stand for too long but what if somebody has to load 4 gels and has them in the same chamber? It is impossible to be that fast in my opinion.
Maroulis on Thu Jul 9 12:13:48 2015 said:
I also realize that it is important to not let the samples stand for too long but what if somebody has to load 4 gels and has them in the same chamber? It is impossible to be that fast in my opinion.
a few ways to do this:
1) after loading each gel, start the run to get the samples stacked, stop the run, load the next gel,...
2) run the gels in separate chambers
3) just run as many gels as you can handle at one time until you get faster (which may not happen, there is a limit to how fast you can load).