Hi guys, I made a mistake with a western blot. i am probing for two separate molecular weight proteins and therefore cut the membrane. I placed the antibodies on the membranes, only to realise 30 minutes later that I got the antibodies mixed up - I put the one that was meant to go on top at the bottom, and vice versa. Can I simply swap the antibodies and continue, or do I have to strip and re-run the whole membrane again?
Dear Robert
If it were me I would strip, block and then re probe with the correct primary ab
Hi there,
Stripping is safer but if you have already shown that both antibodies are higly specific and react only with the protein they have been raised against (and don't react elsewere on the entire membrane) then swapping should be OK.
Thank you for your answers. I ended up just replacing the antibodies and the blots actually came out very nicely. I suspect it's as the molecular weights of the specific proteins were vastly different, and the antibodies were both suspended in the same media (TBS-T). Next time I will be sure not to make the same mistake again!
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