Hi, is it possible to reprobe a blot after visualizing it (by adding another primary antibody, secondary, ECL plus) without stripping it first? I was told by a colleague that I can can skip the stripping process by washing the blot with TBST then re-probe with a loading control (beta actin). I was also told that it is not necessary to strip a blot unless the antibody that I'd be using for the reprobing will compete for the epitopes on the antigen with the first primary antibody.
Please enlighten me in this, thanks.
Dear Elena,
I am afraid you are told wrong. Please strip the blot before reprobing.
I agree with Mukul. However, if it is about detecting different molecular weight proteins then the membrane can be probed with multiple antibodies (targeting different proteins) simultaneously. It is just like we use antiserum to detect different proteins of a pathogen.
If your second probe is for detecting a high-concentration protein, like actin, and there are no overlapping bands, I wouldn't worry so much about having to strip
After all, one can even combine primary antibody incubations after some optimization, such as FOXO-1 and FOXO-3, or MLC1 and MLC3. As long as you're familiar with the antibodies, their non-specific bands and the typical time needed to acquire a good signal, there's no real reason why it's not possible.
Hi Elena,
Yes, I think you can avoid the stripping process and use a new antibody on the same membrane. I have done many times and it worked. Of course, as some other people have already mentioned, you need to be sure that the protein you want to detect does not have the same molecular weight as the previous one, so in case you also have the signal from the previous blot you can distinguish them. If the new antibody has different secondary antibody, that will help you to avoid the detection of previous bands.
When I reprobe a membrane with a new antibody, I usually do the following:
1. Wash the membrane with distilled water
2. Wash two times (10 min each wash) with PBS-Tween
3. Blocking for one hour
4. Add new antibody for over-night incubation.
I hope this helps
Juanma
Hi
I have done reprobing many times without stripping but only if the molecular weights of the two proteins are far eniough on the membrane depending of course on the type of gel that you are using and the percentage since some gels separate middle size proteins and some separate high lolecular weight proteins better.
Also, another important point is the source of the two antibodfies. I try to use a rabbit source for the primary antibody and a mouse source for the beta actin so that I skip the stripping stage since the anti-mouse secondary will not pick the first one if washed well to wash the previous anti-rabbit secondary antibody.
Secondary antibodies can be washed off with TBST but primary antibodies need stripping to wash off.
I think the important questions in this case are:
1) Does the primary antibody you already probed with have any nonspecific bands that show up around the same molecular weight as the loading control you are using?
2) Were the primary antibodies produced in the same animal?
If the answer to either or both of these questions is no, then you should be okay.
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining