Dear All,
I have read many questions and answers in this website, and found out that some people mentioned a method to reprobe another primary antibody of different target protein without stripping, but didn't mention the detail. This is very helpful, since I have bad expierience with stripping, and if two target proteins' molecular weight are too closed to cut a membrane.
Could you kindly give me a detail how to do it? for example, Do I need to block the membrane again or just incubate with another primary antibody? Must the primary antibodies be different species for this method?
Thank you
Hi,
If you are using HRP antibodies, you can bleach the membrane with 30% H2O2 for 20 minutes. We do this for our streptavidin blots since the association is so strong between biotin and streptavidin, but this will work for any HRP-conjugated antibody to completely remove the signal. However, the primary antibodies should still be different species. You won't need to block again after bleaching, although you will definitely want to do a couple washes to remove residual H2O2. If you want to be extra thorough, you can put ECL on the blot and image real quick to make sure the blot is fully bleached, but I've never had a problem. And even if your first antibody binds to a specific protein, after bleaching you can probe with another antibody against a different epitope of the same protein and you should be able to see signal.
Hope this helps!
Asa,
For this to work without stripping three main conditions should be met:
1) the first primary antibody that you use must be raised in a different animal than the second that you plan to use. For instance your first antibody could be raised in mouse and your second antibody should be raised in something other than mouse.
2) the two bands should be far enough apart that residual signal from the first band can be distinguished for the signal of the second antibody used.
3) It helps if at least the second antibody is monoclonal and specific. (Beta actin is a prime example using this method)
You do not need to re-block, you simply need to wash the membrane several times before incubating in primary antibody again in order to remove the ECL from your previous round of blotting.
Finally of note is the fact that if you do incubate with another primary antibody without stripping, there is no way to ensure that that aliquot of antibody will not be contaminated with either the previous secondary or primary antibody that you used (although this has rarely, if ever, been a problem for me).
If you use fluorescent secondary antibodies, you can try to separate the two by using antibodies from different species. If the bands are too close, then you could try adjusting the amount of acrylamide in your gel to get better separation. If the antibodies stick to each other instead of the targets, then you need different antibodies.
In general, stripping causes enough problems, but reprobing without stripping causes more problems (as Andrew pointed out; while it hasn't been an issue for him, that has been an issue for me). If you can, probe with both primary antibodies at the same time and avoid contamination issues.
In general, you can wash your blot with large volume (>200 ml) of TBS + Tween 20 (0.1%) for 2-3 hours at RT and then start using your other antibody. Re-blocking for a short time might help. I have used this approach with antibodies against pERK followed by ERK (same sizes) without problem.
If you share the stripping protocol you are using I might be able to help too.
Add pyronin to your sample buffer for electrophoresis and after blotting well are so clear that you can cut 2 or 3 strips without any problem.
Probe first with the weaker antibody that requires a longer exposure time (~1 or more minutes exposure time). Second probe with the stronger antibody that requires only seconds for a quality signal. It should be easy to discriminate between the two signals on the second blot. I am assuming that you are using chemiluminescence with CCD camera to capture the image. Run each antibody independently as well so that you are assured there is no cross reactivity between the antibodies.
Hi,
If you are using HRP antibodies, you can bleach the membrane with 30% H2O2 for 20 minutes. We do this for our streptavidin blots since the association is so strong between biotin and streptavidin, but this will work for any HRP-conjugated antibody to completely remove the signal. However, the primary antibodies should still be different species. You won't need to block again after bleaching, although you will definitely want to do a couple washes to remove residual H2O2. If you want to be extra thorough, you can put ECL on the blot and image real quick to make sure the blot is fully bleached, but I've never had a problem. And even if your first antibody binds to a specific protein, after bleaching you can probe with another antibody against a different epitope of the same protein and you should be able to see signal.
Hope this helps!
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