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Guide to antibody dilution, including techniques to optimize antibody concentration and suggested dilutions.
Updated May 10, 2022.
When using an antibody for the first time, you may need to optimize its dilution for your specific application and experimental setup. Here we describe how to define the optimal antibody concentration by titration and provide suggested dilutions for antibodies without recommended dilution on the datasheet.
The rate of binding between antibody and antigen – affinity constant – can be affected by temperature, pH, and buffer constituents. Varying the relative concentrations of an antibody and an antigen solution can also control the extent of antibody-antigen complex formation. As it is not usually possible to change the antigen concentration, the optimal working concentration of each antibody must be determined with dilutions for each application and set of experimental conditions.
Usually, antibodies have recommended dilutions for various applications included in the datasheet. However, they may require some optimization in your specific experimental setup.
Search primary antibodies.
The optimal antibody concentration, which gives the best staining with minimum background, must be determined experimentally for each assay and is usually determined using a series of dilutions in a titration experiment. For example, if a product datasheet suggests using a 1:200 dilution, it is recommended to make dilutions of 1:50, 1:100, 1:200, 1:400 and 1:500.
A titration experiment is done by first selecting a fixed incubation time and then a series of experimental dilutions of the antibody. Each dilution should be tested on the same sample type to keep the same experimental conditions.
Many antibodies will have similar batch-to-batch consistency; therefore, only one titration experiment is required in most cases. However, especially for polyclonal antibodies, when there is a change in the staining results between batches of the same antibody, we recommend performing another titration experiment.
Unpurified antibody preparations differ significantly in antibody concentration. If the specific antibody concentration of a given unpurified antibody preparation is unknown, we recommend using a concentration/purification kit and refer to the table below as a guideline.
This table provides various dilutions to use in each application for different antibody formats:
|Tissue culture supernatant||Ascites||Whole antiserum||Purified antibody|
|WB/dot blot||1/100||1/1000||1/500||1 µg/mL|
|IHC/ICC||Neat –1/10||1/100||1/50–1/100||5 µg/mL|
|FACS/Flow cytometry||1/100||1/1000||1/500||1 µg/mL|
|Approximate IgG concentration estimate||1–3 mg/mL||5–10 mg/mL||1–10 mg/mL||-|
EIA=enzyme immunoassays, FACS=fluorescence-activated cell sorting, ICC=immunocytochemistry, IHC=immunohistochemistry, IP=immunoprecipitation, WB=western blot.
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