I am running western Blots for long time. Lately I am facing a new problem. My protein samples seem to degrade on the running gel or in the transfer process. The pre-staining protein marker that I use is being transfer nicely to the PVDF blotting membrane but my samples seem like smears through the lanes (when I transiently stained).
I thought that it might be the lysis buffer or the protease and phosphatase inhibitors that I am using, so I changed all my buffers and the inhibitor cocktails to new ones.
Could it be something in the polymerization of the running gel although I see the leader proteins very well both in the running gel and then in the blotting membrane.
Is anyone have an idea what could be the problem?