Choosing the right secondary antibody for your assay is easy, right? You go to the fridge and pick out the one everyone else uses. Well secondary antibodies are specific too, so this may not always prove successful. What’s more the secondary you are currently using may one day be discontinued. So it’s useful to understand how to choose the right secondary. A secondary antibody functions to recognise your primary antibody in order to bring the attached enzyme or dye to the correct location. To make sure your secondary recognises your primary antibody there are a few facts to keep in mind.
Species specific
Primary antibodies are made in animals or animal-derived cell lines, with antibodies produced in species as varied as mice, rats, rabbits, chickens, goats and donkeys. You need to know the species specificity of your primary antibody and make sure you purchase a secondary antibody targeted to the species of your primary. If the primary antibody is a monoclonal, you need an anti-mouse, if your primary antibody was raised in a rabbit you need an anti-rabbit secondary. One of the key benefits of Affimer technology is that they can be specifically targeted to any species. We have a range of anti-immunoglobulins available, from anti-mouse and anti-rabbit to anti-chicken.
Different Classes
Mammals make five different types of antibodies called classes (or isotypes): IgM, IgG, IgA, IgE and IgD. And not to forget chickens, which make a novel IgY class of antibody. The class is determined by the type of heavy chain in the molecule. Antibodies contain two heavy chains which together form their basic Y structure. IgG antibodies contain gamma chains, IgM antibodies mu chains etc. It is also important to recognise the subclass of your antibody IgG1, IgG2a, IgG2b are all antigenically distinct. In addition to this, antibodies possess two light chains, of which there are kappa and lambda varieties. However, any antibody can only possess one type of light chain, so lambda or kappa chains, which makes things a little more simple. Secondary antibodies to each of these antibody chains can be purchased, allowing more specific recognition of your target primary antibody and giving you the opportunity to use multiplex assays, simultaneously hunting for different antibody types at once to target many different protein types. Yet despite their much hyped specificity, secondary antibodies do sometimes cross-react between species and if your experiment involves double-labelling or multiplexing this can cause problems. As Affimer reagents are engineered and produced in vitro, we are able to generate Affimer binders for specific classes and subclasses of antibody, and ensure that they do not cross react, taking all the hassle out of your selection of affinity reagent for both the simple and complex assays.
Functional tags
Secondary antibodies are often tagged with an enzyme, such as an HRP for use in western blots, a dye, such as FITC for fluorescent visualisation, or a linker molecule, such as biotin, which can be used to purify proteins from a lysate or increase the detection of proteins that are expressed at low levels. Affimer reagents tagged with biotin are available in our catalogue, but should you want anything else adding to your Affimer or want a more specific Affimer, our custom service takes just seven weeks. As all our Affimer products are produced entirely in vitro we don’t use any animals in the process and there’s no risk of any being discontinued, as batch-to-batch reproducibility is assured.