hello

The addition of methanol to a western blot transfer buffer has been suggested to prevent gel swelling during transfer and improve the efficiency of protein adsorption onto the membrane. Many laboratories no longer use methanol since they have found that, for their protein samples, the addition of methanol does not markedly increase efficiency, and omitting it eliminates the problem of disposing of toxic methanol-containing solutions. hope this helps.

regards

Hello Rj Jakie

Rinsing the membrane in methanol for 10 secs is too less a time for anything wrong to happen.

Usually PVDF membrane is activated by wetting the membrane with methanol for a few secs and then equilibrating with transfer buffer for a few minutes.

You have given three washes with PBST for 15 mins each which is quite sufficient. If I were you I would have gone ahead with secondary antibody incubation without hesitation and seen for myself what actually would have happened.

I would suggest you don't re-probe with primary antibody again.

Good Luck.

You can always strip and reprobe, but why not do both and report your finding here. You may find a diminished signal with methanol exposure, or it may be equal. Sometimes the small technical "accidents" can lead to useful information, and therefore it is not always a mistake. Good luck! ;)