Optimized Antibody Dilution Through Titration

How to Choose Antibody Dilutions

This article is meant to help choose appropriate antibody dilutions and offer an overview of methods used to optimize the concentration of antibodies.

The rate of antibody-antigen binding, and the affinity constant, varies with the pH, buffer composition and temperature. By changing the concentrations of the antigen and antibody with respect to each other, it is possible to modulate the degree to which antigen-antibody complexes are formed.

Antigen concentration is difficult to alter in most experiments, and therefore it becomes necessary to find the most appropriate antibody concentration for each antibody, under different working parameters and for different applications, using dilution techniques.

Many Abcam antibodies already list the dilutions recommended for a range of applications on the accompanying datasheet, but these are not absolute and may need to be optimized individually.

Titration to Achieve Optimized Antibody Dilution

In each experiment, the antibody concentration that will achieve the best staining with the least background coloration has to be found. For this purpose, dilution series are used with a titration setup. Thus, if the recommended dilution in the datasheet is 1:200, dilutions beginning from 1:50 should be used through 1:100, 1:200 and 1:400 to 1:500.

To perform a titration, the fixed incubation time is first chosen, followed by an antibody dilution series. The same sample type should be used for each dilution to prevent any change in the experimental parameters.

Since antibodies typically display consistency from batch to batch, a single titration experiment is enough. However, when polyclonal antibodies are selected, the staining outcomes differ between different batches and therefore, titration experiments should be repeated.

What Dilution Should be Used if the Datasheet Does not Suggest Any Dilution?

Antibody preparations which have not been purified have significant variability in the concentration of the antibody. Thus if such a preparation is used, and the antibody concentration is not known, a concentration/purification kit should be used using the following table as a reference:

This table gives a range of dilutions for different applications with antibodies from a variety of sources:

  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
EIA/ELISA 1/1000 1/10000 1/500 0.1 µg/mL
FACS/Flow cytometry 1/100 1/1000 1/500 1 µg/mL
IP - 1/100 1/50–1/100 1–10 µg/mL
Approximate IgG concentration estimate 1–3 mg/mL 5–10 mg/mL 1–10 mg/mL -

About Abcam

Abcam is a global life sciences company providing highly validated antibodies and other binders and assays to the research and clinical communities to help advance the understanding of biology and causes of disease.

Abcam’s mission is to serve life scientists to help them achieve their mission faster by listening to their needs, continuously innovating and improving and by giving them the tools, data and experience they want. Abcam’s ambition is to become the most influential life science company for researchers worldwide.


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Last updated: Jun 6, 2019 at 12:49 PM

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