WB anti-caveoline1 not reproducible - (Nov/27/2007 )
I´m having serius trouble in optimizing a WB for caveolin 1: some times I have a band with the expected MW (22KDa), and a band of higher MW (200 KDa aprox) near the top of the gel, but other times I have almost only the band of higher MW. I´m using the standar Laemli conditions and I´m boling for 10 min, and using 1% betamercaptoethanol. Haw can I know if the problem is during SDS-PAGE or during transfer or else??????. This is a previusly reported WB (many reports), with no special protocols mentioned.
try variations in transfer conditions. some conditions suit higher mw than lower Mw proteins.
10 minutes is a long time to boil the samples. 5 minutes should be more than enough (unless you are at 65C, then 30 minutes would be good).