Western Blot: Re-use of membrane? - (Sep/22/2007 )

Hello. I've done my western but so unfortunately during the developing process, our kodak machine broke down. so, i decided to keep the membrane although I'm not sure the correct way to do this (or is this possible anyway??). But what i have done: I washed the membrane again for a few minutes and keep it soaking in PBST at 4C over the weekend. Now, I'm just wondering wheater or not I will be able to use the same membrane straight away to view the results? or should i repeat any steps like blocking or incubation with ab or developing it again before i can see my results or my membrane has done no good at all? For information, I diluted my ab in blocking buffer and this ab is conjugated together with its primary ab (anti-His (c-term) HRP).

Can someone be kind enough to teach me the proper way to this. Thanks

Frustrated spices

Hello. I've done my western but so unfortunately during the developing process, our kodak machine broke down. so, i decided to keep the membrane although I'm not sure the correct way to do this (or is this possible anyway??). But what i have done: I washed the membrane again for a few minutes and keep it soaking in PBST at 4C over the weekend. Now, I'm just wondering wheater or not I will be able to use the same membrane straight away to view the results? or should i repeat any steps like blocking or incubation with ab or developing it again before i can see my results or my membrane has done no good at all? For information, I diluted my ab in blocking buffer and this ab is conjugated together with its primary ab (anti-His (c-term) HRP).

Can someone be kind enough to teach me the proper way to this. Thanks

Frustrated spices

I agree, the enzyme activity is probably dead by now. Best bet is to reincubate in secondary antibody. There are usually plenty of unoccupied epitopes on your primary so you should be able to bind enough fresh secondary to get a signal. Then reapply substrate and hope that your developer is working today Should not be necessary to reblock the blot.

BTW, you might consider fluorescent Westerns with fluorophore-labeled secondaries. Bypasses the developer frustrations, and the signal lasts basically forever. I really love 'em.

Hi
Agree with both posts.
HRP conjugated to anti-his Ab is already enzymatically dead.
But you can try reprobe with HRP conjugated secondary Ab anti to the source of your primary HRP conjugated Ab.

E.g. if you used mouse HRP anti-His, then reprobe HRP conjugated anti-mouse.

NOTE: I haven't tried it before though and not sure if the conjugated HRP will interfere with the binding. Good luck.

...-...

Thanks a lot for the advice & sorry that this come quite late.
Thanks again.