The optimal antibody concentration, or optimal working dilution, is the one that gives the best staining with the minimum background; this must be determined experimentally for each assay.

Many of our antibodies have dilution factors included on the datasheet under the 'Applications with Dilution' section. However, these can require some optimization, as we describe in this article. Varying the relative concentrations of an antibody and an antigen solution can control the extent of antibody-antigen complex formation. As it is not usually possible to change the concentration of the antigen, the optimal antibody concentration must be determined for each application and set of experimental conditions.

How do I optimize the amount of antibody to use from a given dilution factor?

The optimal concentration is usually determined using a series of dilutions in a titration experiment.

A titration experiment is done by first selecting a fixed incubation time, and then a series of experimental dilutions for the antibody. If a product datasheet suggests using a 1:200 dilution, it is recommended to test dilutions of 1:50, 1:100, 1:200, 1:400 and 1:500. Test each of these chosen concentrations on the same sample type to determine which one works best.

Many antibodies will have a similar batch-to-batch consistency, so in most cases, only one titration experiment is required. However, if there is a change in the results of the staining between batches of the same antibody, we recommend performing another titration experiment. Such variation may be more common with polyclonal antibodies.

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