I'm interested in incubating my membranes with several antibodies and wonder how many incubations a membrane can undergo before having too much background and non-specific binding. Usually I incubate my nitrocellulose membranes once and then, after revelation, I use a stripping step to incubate the membrane with the loading control antibody.
Why don't you use several blots and treat them independently? Using the same blot and striping it is not a good idea. Most of the time stripping blots doesn't give a health report, and you will lose a lot more time by doing this as compared to another way around
it really depends on what you use for revelation, otherwise I join Mahesh Chandak opinion its not reliable
To avoid high background after reusing a membrane for further detections, always start with an additional round of blocking. Personally, I try to avoid stripping at all, because you'll always lose your protein of interest to some extent. For detection of several proteins on the same membrane, you should start with the one that requires the highest sensitivity for detection. You could then either detect one after another (if molecular weights differ enough), cut the membrane and use each strip separately in parallel or cover the parts of ther membrane during the detection, where you don't expect your protein of interest. All of these approaches worked fine for me in the past.
Agreed with @carsten above. I never strip, as it can affect signal from your protein of interest. I usually get away with about 5 probes per membrane. For order, i usually do the cleaner (fewest bands) or most sensitive antibodies first, and the strongest ones last. I usually re-block between probes, at least through the first 3. For each new probe, just be very careful about which bands are truly new and were not present before (take your previous exposures with you to the developer/imager). If a probe doesn't work, the membrane might be too blocked up but I've found that re-activating the membrane with methanol (just 20 min incubation or so) can help. Good luck!
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