How to choose an isotype control antibody

Your guide to selecting the right isotype control for your experiment.

Updated October 18, 2022

Contents

• How to choose isotype controls
• Different types of isotype controls
• Popular isotype control products

An isotype control is an antibody selected as a negative control for flow cytometry experiments and is used to detect non-specific binding. Isotype controls can be conjugated or unconjugated and should match the host species, the Ig subclass, and the conjugation of the primary antibody used against the target of interest. For example, a rabbit monoclonal to CD4 FITC-conjugated should be matched with a rabbit IgG isotype control FITC-conjugated.

An isotype control will determine the presence and extent of non-specific antibody binding, which arises when an antibody binds to endogenous Fc receptors or interacts with off-target proteins and molecules. Choosing the correct isotype control for your experimental conditions is crucial to distinguishing the true antibody signal from non-specific background staining, as well as to limit off-target interactions with dyes or enzymes conjugated to the antibody. 

Browse our total range of isotype controls

How to choose your isotype control

Optional: Make sure isotype controls are suitable for your experiment

Isotype controls are suitable for many applications either as a negative control (Flow cyt, imaging) or as a standard blocking and protein coating agent (IF, IHC, WB, ELISA). As isotype controls were developed for surface staining, they generally do not work well with intracellular staining. Most importantly, these controls should not be used to differentiate positive and negative cells or to set positive gating regions.

1: Choose an isotype control that does not recognize your target antibody.

To effectively determine non-specific binding, isotype control antibodies must be specific to a target, which is not present in your sample. 

2: Choose an isotype control raised in the same host species as your target antibody

Both the host and class of the isotype control antibody impact how it interacts with Fc and other cell components in your sample.

3: Choose an isotype control that matches the class and sub-class of your primary antibody.

As the five different heavy chain classes (also known as isotypes) have different functions and locations, it’s important your isotype control antibody matches that of your primary antibody. While class and sub-class may not need to be matched for isotype controls used in western blot or IP, this is important in flow cytometry and double-labeling experiments.

You can find the isotype of all primary antibodies under the ‘Properties’ section on our datasheets.  

Check the class and sub-class of our isotype controls in the table below.

Optional: Minimize experimental differences

• Manufacturing process – primary and isotype control antibodies from the same supplier tend to work best together.
• Clonality –  whether your primary antibody is polyclonal or monoclonal, make sure your isotype control has the same clonality.
• Format and purity – both can vary depending on supplier or intended use for the antibody. What the exact purity and format of your primary is not important, just make sure the isotype control matches it.
• Volume - if using test antibodies, identify the microgram quantity of the antibody at that volume and get an equal mass of isotype control.
• Concentration – like format and purity, concentration can vary across different antibodies and suppliers. If you have optimized your primary antibody dilution, do the same for your isotype control.
• Storage and handling conditions - if using a secondary antibody, add the isotype control in the same way as your primary antibody.

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Different types of isotype controls

There are five main mammalian antibody isotypes: IgA, IgD, IgE, IgG, and IgM. IgG isotypes can also be further divided based on light chains. Different species have different sets of isotypes, for example, rabbits have more IgG while mice have more IgM. IgG isotypes are more specific, so antibodies produced in rabbits have higher specificity and affinity.

Use the table and links to select the isotype control matching your primary antibody. You can then filter product lists using the 'Conjugate' search filter on the left-hand sidebar to find products that match your conjugate.