In order to perform an antibody titration, I have found many different protocols with different recommendations, but most of them suggest to do dilutions in volume without taking the concentration of the antibody into consideration (for instance, 1uL of antibody into 49uL of PBS). As the concentration of the antibody is going to be different in each vial of product, it is also recommended to perform a titration every time a new antibody is purchased, even with the same order number, clone and so on. Does it make more sense to proceed with dilutions considering concentration to avoid infinite titration procedures?
Another question for me: when performing a new experiment with different antibodies that will coexist in the same tube, is it needed to perform a combined titration of both Abs at the same time?
The most common works on antibody titration is depending on the volume dilusion..
You are right
It is always more accurate to work with the concentration. The problem is that this is only possible when the Ab is a highly purified monoclonal Ab (no other proteins as the ones used for revealing the reaction or Abs must be present in the sample). That is the reason why dilution is used instead of concentration when working with serum, culture supernatants, polyclonal Abs and even in-house or commercial conjugates. This is also why, in those cases, the working dilution of every stock must be previosly standardized.
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