are you trying to see which antibody is more effective? If you just wash ecl off and use other 2ndary antibody, all you might get is a dirtier blot, indicating non-specific binding. I would suggest doing a gentle stripping of the membrane ( you can look up protocols online), which will strip primary with secondary, re-incubate with primary and use the other secondary of interest. The signal you get might be NOT as strong as the first time you put ECL on the membrane, its because stripping of the membrane not only takes off primary and secondary, but might take some protein away as well. I think this is the only thing that will work if you are trying to decide between the two secondaries.

Hope this helps :)

If the first secondary antibody is polyclonal it most likely will not work. If the first secondary is monoclonal and second is polyclonal than there is a chance.

With both secondary being monoclonal it's more complicated. It depends if antibodies epitopes overlap or sterically interfere in some other way with the binding of each other. Usually the pairs of two monoclonals are tested prior to this experiment by using Biacore, etc.

Also keep in mind that ECL reagent contains peroxide and this oxidizes the whole sandwich from top to bottom. Thus binding on all levels gets much worse and reactive radicals formed will also interfere with the next ECL reaction.

We were trying to find a way to regenerate proteins for consistent second probing.

Something like ethanolamine or ascorbic acid might help but not 100% effective.

So it's easier to probe again.

If as Yaruna suggested you're trying to compare antibodies you can try to do competition, i.e. one secondary labelled mix with another unlabelled secondary in different proportions. Otherwise this experiment is easier done with ELISA or Biacore.

Regards,

Galina