Which dilution to use
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 antibody and antigen in solution can also control the extent of antibody-antigen complex formation. As it is not usually possible to change the concentration of antigen, therefore, the optimal working concentration (dilution) of each individual antibody must be determined for each application and set of experimental conditions.
Many Abcam antibodies have recommended dilutions for various applications included on the datasheet. We recommend trying these dilutions to start with. However, they may require some optimization.
Optimizing the antibody dilution: titration experiments
The optimum titer or dilution is the concentration (dilution) which gives the best staining with minimum background / non specific binding. The optimal antibody concentration 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 optimal results. For example, if a product data sheet suggests using a 1:200 dilution, one may wish 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. Then a series of experimental dilutions of the antibody is prepared ready to test. Each dilution should be tested on the same type of sample, to keep the experimental conditions standardized.
Many antibodies will not vary much in performance from batch to batch, and you should only need to perform one titration experiment. However, polyclonal anti-sera antibody concentrations can be significantly different from animal to animal, or from one serum bleed to the next. In these circumstances, or when you notice a change in the results of the staining between batches of any antibody, we recommend performing another titration experiment to obtain the optimal concentration for that vial.
Suggested dilutions for antibodies with no recommended dilution on the datasheet:
Unpurified antibody preparations vary significantly in specific antibody concentration. If the specific antibody concentration of a given unpurified antibody preparation is unknown, one may refer to the following "typical ranges" as a guideline for estimation:
This table provides a guideline dilution to use for each application when using various formats of antibody: