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!!