Blotting the same membrane for two different protein saves the time and cost. There are many ways of doing this either with stripping or without stripping. Which is the better approach and can we mix the specific monoclonal antibodies together for two different proteins on the same membrane without stripping?
In my opinion, it depends on what you are aiming for. If you want to quantify the protein expression, then the best way is to have the protein of interest and loading control on the same blot. Alternately, you can cut the membrane and develop them together with same exposure time. Stripping and reprobing is not recommended for quantitative analysis.
As everybody saying cutting the membrane is the best way forward. Principally, if the antibodies dont cross react at all and if the proteins can be distinguishable clearly as two different bands on SDS-PAGE, you can probe them together.
We used to incubate the membrane with several antibodies (I think 5 or 6 at most). Every over-night incubation we incubated with another one, without stripping in between. It works perfectly if the proteins of interest are of different size so you can differentiate them on the blot. Of course you will also see some of the other antibodies when developing but is that a problem for you? Normally they wont disturb you because you know what size you have to look for. I never tried an antibody-mix but why not? It' only a bad idea if you want to reuse the diluted antibody because you have them in a mixture everytime then. And of cause you need to know the individual optimal concentrations of the antibodies.
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