2. Validate your antibody before first use and when you observe lot-to-lot variability
As a result of the production process, the clonality of the antibody may dictate its reproducibility.
Antibodies are produced when a host animal is challenged with the target antigen. Serum from that host contains polyclonal antibodies produced by a number of different B cells, each of which raises an antibody to recognize a different epitope on the antigen. Single B cells can also be isolated from the host’s spleen and fused to myeloma cells to create hybridomas—hybrid cells capable of secreting monoclonal antibodies specific to a single epitope.
Since they originate from a single cell that is cloned and cultured, monoclonal antibodies tend to exhibit minimal lot-to-lot variation. In contrast, each lot of polyclonal antibodies is produced by a new host immunization. Due to the nature of the mammalian immune system, each immunization will yield a slightly different arsenal of antibodies, which affects lot-to-lot reproducibility.
One should also consider the purification method used, as some methods may leave behind impurities that could affect results for more sensitive techniques. For polyclonal antibodies, Protein A or Protein G purification yields a less homogenous product than immunogen affinity. It is best to verify in advance the level of purity required and use antibodies purified in the same manner when repeating a set of experiments.
For these reasons, it is important to validate every lot of material for your application. If a new lot fails to perform comparably to a previous lot, reach out to the vendor to see if they have made any changes to the antibody and ask for technical advice, if necessary. Discard the lot when appropriate (see #3).
The first steps of antibody validation are to perform a Western blot to verify that the antibody recognizes a protein of the expected molecular weight and then use immunocytochemistry or immunohistochemistry to verify the correct subcellular localization. Use a panel of positive and negative cell lines or tissues with variable expression levels of the protein of interest. If such lines do not exist, transfect the protein of interest in nonexpressing cells to create a positive control or use RNAi to knock down the protein of interest to generate a negative control.
In addition to validation by Western blot, the antibody must also be validated for any end application, whether it is ELISA, immunoprecipitation, or another technique.