Hello, everyone. I am running SDS_PAGE gel and while running the gel for about 10 minutes, it seems like the sample wells are getting diffused with the blue colour (of the sample loading buffer). What could be the reason? Is there a problem in the voltage or current? Or anything else?
Thank you.
(I am attaching the SDS-PAGE gel pictures.)
I think the reason may be the density of the loading buffer. Try with fresh/new loading buffer/dye.
This is nothing to worry about. The bromophenol blue is a small molecule and will diffuse relatively quickly. The proteins will be running perfectly, but you’ll only see that when you stain the gel.
As long as you get the sample cleanly into the very bottom of the well (don’t let the sample sink to the bottom) everything should be fine.
This could be a wrong relation between the sample and the loading buffer. I use 4x at a final concentration 1X. If that doesn't change this, maybe your adding too much sample. The quantity of wells have a maximum of volume capacity which decreases as the number of wells increases. I don't think you have a buffer or voltage problem, if so your marker should not be running and in the images you can see the movement. In any case, I would recommend you to try a low voltage, 80-90 V constant to ensure an even run just to be sure.
Macarena Echeverría-Bugueño Thank you so much for your answer. I resolved the problem by mixing the 5X loading buffer (final concentration 1X, as yours) with my samples and denaturing them, and then spinning shortly and it worked.
sometime can occurs because the volume of well is full, but its dont problem, in my experience.