Optimal Concentration of the Antibody

After having purchased a new antibody, it is necessary to determine its optimal working concentration before putting it into the experiment. This is a task that confronts all immunohistochemists, often being the main reason for frustrating results (or perpetual mediocrity) experienced by many laboratories. This predicament is described in more detail by Miller (2001). 

Purified antibodies are usually prepared from whole serum by affinity chromatography. Concentration of affinity-purified specific antibodies is normally specified by the manufacturer in the antibody data sheet. When starting with a new 

The optimal working concentration can be determined in a series of stains with the antibody in several dilutions of twofold increments (e.g., 1 25, 1 50, 1 100, 1 200, 1 400, 1 800, 1 1600). The resulting antibody concentration can be calculated according to the following formula. 

Note Final volume may be approximated as the volume of the antibody diluent in case of high dilutions. For low dilutions (les than 1 10), the final volume is calculated as follows final volume = volume of antibody required + volume of antibody diluent. 

The best stain is then the optimal for that particular primary antibody if you do not observe any nonspecific background staining in control incubations (see below). 

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