Protocols

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Isotype Control Antibody Selection Guide

I. Introduction

The selection of an appropriate isotype control is essential for every flow cytometry experiment. Isotype control Abs that have no relevant specificity help to distinguish non-specific "background" staining from specific antibody staining. There are three main factors that contribute to the levels of background staining associated with a primary Ab: 1) binding to Fc receptors on target cells, 2) non-specific protein interactions with cellular proteins, lipids, or carbohydrates, and 3) cell autofluorescence. All of these factors can greatly vary depending on the target cell type and the isotype of the primary Ab. Thus, isotype controls need to be matched to the specific primary Abs (species and isotype, including heavy and light chains) being used in order to accurately determine the level of specific staining by the primary Ab. The most common monoclonal Ab isotypes are IgG1, IgG2a, IgG2b, IgG3, IgM, and IgA. Recommended isotype controls can often be found on the Technical Data Sheets for primary antibodies.

II. Finding an appropriate isotype control

A. Determine the isotype of your primary Ab. Skip to Step B if you already know the isotype and origin species of your primary Ab.

1. Search for your Ab at www.bdbiosciences.com by entering your target antigen, a clone name, or a known catalog number. *If you are interested in a particular fluorescent tag or conjugate, type that into the search box along with your target antigen or clone name (biotin, FITC, PE, APC, PE-Cy5, PE-Cy7, PerCP, Alexa Fluor 488, Alexa Fluor 647, etc.). *Do not type in "anti-" if you are searching for an Ab.
2. Identify the isotype of your Ab and the species of origin in which the Ab was produced, which are listed under the "Isotype" column.

B. Search for your isotype control.

1. Search for your isotype control using the search engine. - Type in the word "isotype", the species from which the Ab is generated (mouse, rat, hamster, etc.), the isotype (IgG1, IgG2a, IgG2b, IgM, etc.), and the fluorochrome (FITC, PE, APC, etc.) separated by spaces.
2. Select an appropriate product from the list. *Be sure to read the Technical Data Sheets for the appropriate application of each isotype control product (for example, surface or intracellular flow cytometry).

III. Using your isotype control

For optimal results, use the isotype control product at the same concentration as your primary Ab.

Please contact Technical Support (1-877-232-8995) if you need assistance with choosing an isotype control.