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Guide to antibody dilution, including techniques to optimize antibody concentration and suggested dilutions.
The rate of binding between antibody and antigen is dependent on the affinity constant. The affinity constant can be affected by temperature, pH and buffer constituents.
Varying the relative concentrations of an antibody and an antigen in solution can also control the extent of antibody-antigen complex formation. As it is not usually possible to change the concentration of the antigen, the optimal working concentration of each individual antibody must be determined, with dilutions, for each application and set of experimental conditions.
Many Abcam antibodies have recommended dilutions for various applications included on the datasheet. Our scientific support recommends trying these dilutions first. However, they may require some optimization.
The optimal antibody concentration, which gives the best staining with minimum background, must be determined experimentally for each assay and it is usually determined by using a series of dilutions in a titration experiment.
When a new antibody is received in the laboratory, it is advisable to try a titration experiment to determine the optimal antibody dilution for best results. 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. This should determine the optimal dilution for your individual laboratory and sample conditions.
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 type of sample in order to keep the same experimental conditions.
Many antibodies will have similar batch-to-batch consistency, therefore in most cases only one titration experiment is required. However, especially for polyclonal antibodies, when there is a change in the results of the staining 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 to use a concentration/purification kit and refer to the table below as a guideline.
This table provides various dilutions to use in each application from different sources of antibody:
|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||-|