Well, my boss is of course livid.
This Western has been an ongoing problem child, but I feel that the cherry on top was last night, when I finished stripping the membrane at 10:30 then decided to start the primary after all.
But, I was so occupied with which protein I should reprobe for, that I completely forgot you need to block after stripping. Which, frankly, had I remembered: I would not have started the primary that night.
Here's my standard procedure:
Strip with 2.5pH glycine, 0.2M for thirty minutes
Wash three times in PBS-T, 10 minutes each
Image to test for residual antibody
Block in 3% BSA in PBST, 1 hour room temp
Incubate in primary overnight
Except I forgot to block. So it went straight into primary antibody after washing. Does anybody know how bad that is, on a scale of 1-10? My boss doesn't, because he apparently doesn't make mistakes, ever. I believe his exact words were "I don't know, because I've never done that." (Followed by "'It was late' is not an excuse!" For a neuroscientist, he certainly doesn't seem to have much appreciation for the effects of exhaustion or anxiety on student function.)
This membrane has already been stripped twice. I'm loath to do it a third time, but not sure I have any choice.
It's not like I wanted to forget...