Interesting question - I am using a lot of phospho-antibodies, some of them diluted in milk, most of them diluted in BSA (according to the companies recommendation), but I block always with milk before, regardless of having the dilution in milk or BSA. And I reuse them till they don't work any more.

I am working with nitrocellulose membrane these days, but long back I was using PVDF membranes, and there this approach also worked fine. I had much worse experience with non-specific bands /background literally looking like a Ponceau staining when doing the blocking, primary and secondary antibody dilution with BSA only, what the antibody supplier abcam suggested for establishment. When I went for my common lab protocol for this antibody (whole protein) using milk only, I could avoid this problems.

And if you take the recommendation of CST (Cell Signaling Technology), in my opinion one of the best antibody selling company, and go through their protocol, they recommend blocking with milk, and primary antibody sometimes with milk, sometimes with BSA, depending on their in house validation.

If you wash the membrane thoroughly after blocking (washing time is not that important, it is more important to completely remove the milk), I don't see any problem to reuse an antibody.

You can cross-check if the company gives any recommendation for the blocking, primary and secondary antibody or call the technical support - if you strictly follow their protocol and fail, normally you will get help from their technical support, otherwise they will advise you to follow their recommendations.

But I don't see any reason why you shouldn't be able to reuse an antibody.

Hi Eduardo,

Yes , you can and you can also follow the comments of Daniela Bruennert stated above. In addition you can first wash your membrane in your buffer ( TBST 1X ) severally before you use the antibodies.

I hope this will help you.

All the best!

yes