I am considering to combine two different primary antibodies to detect different target proteins on the same membrane (Nitrocellulose/PVDF) for western blot. I found that this works but my question is that is it okay/possible to reuse antibody solution both primary and secondary when we combine two different primary antibodies?
Hello Md Nasim Uddin
It all depends on the antibody. It depends on the strength of the signal and stability of the antibody. Also, it depends on the working titer of the antibody and how much of the antibody is consumed during the first use.
Re-using diluted antibody is not recommended because the antibody is less stable after dilution and older dilution buffer is prone to microbial contamination.
However, if you wish to re-use the antibodies, I suggest you may re-use primary antibody but never re-use secondary antibody. Do not dilute antibodies in milk if you wish to re-use them because the milk will go bad and fall out of solution.
BSA is ideal, and so you may dilute your antibodies in 3% BSA in TBST. Bacterial contamination is another concern when you re-use antibodies. So, to avoid microbial contamination, sodium azide (0.1%) may be added as a preservative and used repeatedly until the antibodies start showing weak signal.
Also, avoid repeat freeze-thaw cycles of the antibody solution to be re-used as this may affect the stability of the protein.
Best.
@Malcolm Nobre, I re-use single primary antibody solution for few times. Now I am considering to mix two different primary antibodies as a single solution and I have tested them individually and re-used for few times successfully. I am interested to know if I combine them will there be any issue to re-use them? I do not like to loss them after a single use as cocktail! Any idea about this? Thank you!
Hello Md Nasim Uddin ,
I don't advise you to do so, as you will have more than one antibody consumption, which will decrease the stability even more. Moreover, you have a higher possibility for antibody-antibody interaction. What I advise you, if you are thinking of doing so just to save time, try cutting the membrane at appropriate molecular between the two proteins to be detected and incubating each antibody alone.
Good Luck
Lina
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