I am experiencing streaking of protein lysates that come from mouse spleen tissue on an SDS-PAGE gel (hand cast, not store bought). I know that too much protien is a problem so I tried several dilutions (7 ug is the lowest I loaded) and still no dice. I hear that too much salt is a problem, but I don't know how much is too much? My RIPA buffer that I've used to lyse in is 150mM NaCl. This seems pretty standard, so I'm not sure if this is it. I'm also getting some nasty background on one of the antibodies I use on these samples, and the other one I use is generally clean.
How can you tell protein degredation from streaking caused by too much protein? I've been trying to trouble shoot this for months now. I am trying to validate antibodies on mouse tissue that are knocked out for my protiens of interest (spleen to be exact). I know my antibodies are good on human and mouse cell line lysates.
Any and I mean, ANY suggestions are greatly appreciated. Thanks