Antibody suddenly does not work any longer, what can be the reason?

An antibody losing its activity is an issue that occurs from time to time in each laboratory. How can this be explained?

Antibodies are proteins of varied stability, and this cannot be predicted in advance. Therefore, some antibodies may lose activity following one year of storage, while others can remain active for 20 or 40 years. There is no simple way to control this process.

The following factors can contribute to antibody stability:

Storage temperature
Purity grade
Presence of stabilizers like glycerol, BSA and commercially available formulations
Usage of the same antibody solution multiple times

These factors may influence decreased or lost antibody binding. End users often request another antibody batch, hoping that it is going to perform better. However, if an antibody is prepared from the same source material, testing the other batch may have the same outcome.

Storage temperature: Please read the provided product sheets carefully. If they are not provided, contact the antibody supplier (the company or lab supplying the antibody). The storage temperature differs between antibodies. Some antibodies should be stored at -80°C, while others may require storage at 4-10°C.

Antibody purity grade: For antibodies provided in serum format, there are many stabilizing proteins present beside the antibody itself, which can contribute to a longer shelf-life. However, this antibody format is not suitable for all applications. One should keep in mind not to purify all available serum at once, in case of stability issues with the purified antibodies.

Presence of stabilizers: Examples of these are glycerol, BSA and commercially available formulations. Such stabilizers should not be added to antibodies present in serum format, as these already contains stabilizing proteins. Addition of 25% glycerol (final concentration) will prevent an antibody solution from freezing when stored at -20°C, and may prolong the antibody shelf-life.

Usage of the same antibody solution: For some antibodies, this approach will result in no signal in the second incubation, as all of the primary antibody binds to athetarget protein in the first incubation, and there is no more antibody left to bind.

Antibody suddenly does not work any longer, what can be the reason?

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