Help with western blot!! - (Apr/04/2014 )

I did WB with the same sample, the first time the band is very good, and signal is strong, however, when I take out the sample from -20 degree, and boiled again for another 10 min (when I first extract the protein, I also boiled 10 min), and I use just same primary antibody and same concentration, then I didn't get any band.

Why is this?? anyone has any ideas? I think it is probably the proteins are degraded when I did the second 10 min boiling. Has anyone also has this problem??

Thanks in advance!!

Proteins can aggregate and precipitate out of solution with prolonged boiling.  If you have boiled them once, you don't usually need to boil them again.