SDS-PAGE top-half only background - (Oct/19/2005 )
I am running my protein samples on two 10% SDS-PAGE with 0.25% b-glucan as substrate (using regular Tris-glycine SDS buffer). One gel is used to do a zymogram and the other one is transferred on a membrane for western blot.
After renaturation washes of zymogram, incubation and Congo Red staining of zymogram to see the clearing zones around the proteins, I cannot observe halos for the proteins a the top of the gel because all the top-half of the gel seem not to be stained. For my enzyme control, which run into the lower part of the gel, a halo can be observed as usual.
What is making it even weirder is that, for the western blot, after hybridization and colorimetric detection, everything like usual, the full top part of the membrane is all dark even for the empty lanes and MW (with only some white protein bands in the lanes where samples were loaded). Again, as my enzyme control migrates lower in the gel, it is well detected by my antibodies but not my larger proteins in the top part of the gel.
Therefore, both the zymogram and the western blot seems to have the same problem! I am suspecting the SDS-PAGE running to be the common problem. But what? I cleaned the plates and apparatus, i made fresh running buffer, i have checked the pH of my solutions, used new Bis-acrylamide, did not solved the problem. I regularly use those techniques and they always worked nicely before.
Does anyone has observed this before? Any ideas?
glucan is probably running along with the protein.instead of taking glucan solution while preparing gel, try mixing glucan with tris to be added during resolving gel preparation.
hope this was helpful