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secondary antibodies
Photo courtesy of Steven Depolo.

Everyone likes a little recognition now and then. A little nod from someone that they recognize what we do.

While personal recognition will make you feel good, recognition is key to making sure your Western blots have a signal. Secondary antibodies need to recognize the primary antibody to bring your enzyme/label to the right spot.

Last week we talked about conjugation of your secondary antibody to various labels and enzymes. This week we are telling you about things to consider to make sure your secondary antibody recognizes your primary antibody.

Know your beast

Primary antibodies are made in animals or cells isolated from animals. Mice, rats, rabbits, goats even chickens, it seems no animal can escape the injection of antigens.

You always need to know the species specificity (the animal species in which it was made) of your primary antibody. And you need to buy a secondary antibody specific for the species.

You have a monoclonal primary antibody? It was probably made in mouse hybridomas – buy an anti-mouse secondary. Your primary antibody came from the chicken farm – buy an anti-chicken antibody.

Have some class

Mammals make five different types of antibodies called classes (or isotypes): IgM, IgG, IgA, IgE and IgD. They can be produced at different times during the immune response or at different places in the body. The class designation is based on which protein is used for the larger proteins in the antibody complex (called the heavy chains. For a complete description of the antibody molecule, see the Advansta WiKi article on Secondary Antibodies). IgG molecules contain gamma chains, IgM mu chains, and so on.

To make it even more confusing, there are slight differences in some heavy chain molecules necessitating a subclass terminology. IgG1 antibodies have a gamma chain that is antigenically distinct from an IgG2a gamma chain.

Likewise, the smaller proteins in the antibody complex (called the light chains) can be one of two proteins called kappa and lamba. Luckily there are only one type of each of these – phew!

Secondary antibodies can distinctly recognize any of these chains and you can use this to your advantage!

While polyclonal antibodies are usually of the IgG class, containing a mixture of the different subclasses, monoclonal antibodies are made by clonal cell lines and thus only 1 specific IgG molecule is made. You can buy a sub-class specific antibody (e.g. anti-mouse IgG2a) to help you detect multiple mouse primary antibodies of different subclasses simultaneously.

If you don’t know the subclass, or don’t care, you can buy a pan anti-IgG antibody that recognizes all subclasses.

Except if you have a primary antibody made in a bird – chickens are special you know. Birds make a novel antibody molecule called IgY and anti-IgY secondary antibodies must be used when working with primary antibodies produced in chickens or eggs (whichever came first).

Which part are your secondary antibodies recognizing?

Why did I bother bringing up heavy and light chains a little while ago? It wasn’t just to introduce the ideas of class and subclass. You can also purchase secondary antibodies that recognize specific regions of the primary antibody.

Anti-H+L secondary antibodies recognize both the heavy and light chains of the antibody molecules. Since there are only 2 light chains that can be associated with all heavy chains, this is a good antibody to use if you don’t know the class of your primary antibody.

Alternatively, you can buy an antibody that only recognizes the light chain. While not used for most Western blots – they serve an excellent purpose when doing IP-Westerns.

If you do an IP-Western with a secondary antibody that recognizes both heavy and light chains, you are going to find 2 very strong bands on your blot – one at approximately 50kDa and one at 25kDa. These are the heavy and light chains, respectively, of the primary antibody. The signals can be so bright from these bands that they overwhelm the blot and obscure visualization of any proteins that have similar molecular weights.

If you use the anti-light chain secondary antibody, then you will only have to contend with the band at 25kDa.

Think that is all there is to know about choosing a secondary antibody? It isn’t. Come back next week when we talk about different types and purity levels of the secondary antibody.

 

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