Now, we have separated the proteins and transferred them onto a membrane. It’s about time we made them visible, isn’t it?

The detection of the protein of interest is based on the application of a complex of two different antibodies. After the transfer, the membrane is incubated with a primary antibody that recognises the specific protein but is not visible in itself. 

In order to detect the primary antibody, a secondary antibody, directed against the primary antibody, is added. The secondary antibody is linked to a conjugate, for instance an enzyme, which reacts with the developmental agent or catalyses a reaction which produces a final product which is detectable, for instance by light emission or colour. 

 

Principles of antibody application and detection in Western blotting. A) Membrane to which the protein has been transferred. B) Primary antibody is applied. C) Secondary antibody linked to a conjugate is applied. D) Detection reagents are added and after development, the conjugate which marks the place of the protein, is visible.

 
Obviously, you should choose an antibody that is known to be directed towards the protein you want to detect. Even so, there is always a risk that the antibody may bind to other proteins that have sites that resemblance of your protein of interest. Trying to minimize this risk is therefore a very good idea. 

First, you may try to find a monoclonal primary antibody, rather than a polyclonal. While both types of antibodies have their advantages in different applications, in terms of Western blotting, a monoclonal antibody is preferred. It is considered to be much more specific to the protein of interest than a polyclonal antibody. Monoclonal antibodies are produced from a single cell line, and therefore, react to only one specific epitope. Polyclonal antibodies have a broader antigen spectrum and are excellent for quantitative binding, but not optimal when you are trying to single out one specific protein. 

However, if you incubate the membrane directly with the primary antibody, there is a huge risk that it not only binds to the protein you want to detect, but also to lots and LOTS of other proteins on the membrane. This is called non-specific binding, a.k.a. high background, and makes it hard to discern your specific protein.

 
A blot with high background.
 
What you can do in order to avoid this scenario, is to pre-block your membrane with another protein solution which masks all the free binding sites on the membrane, i.e. all the sites which do not contain blotted proteins from the gel. By doing this, the only places left for the antibodies to bind to, are the places which contain your wanted protein.

This protein solution usually contain fat-free dry milk or bovine serum albumin, along with Tween®20. Tween®20 is a detergent which appears to have a renaturating effect on the proteins, which improves the strength of the antigen-antibody binding. It has also been shown to help decreasing high background. 

Washing is also important. By washing the membrane thoroughly after each antibody application, all unbound antibodies are washed away and are thereby unable to interfere with any free binding site that may still be left on the membrane.

 
Wait a minute… did you say milk?!
 
I did! The proteins that are present in milk have been proven to bind strongly to PVDF and nitrocellulose membranes. Therefore, milk is providing an efficient and also very cheap way of blocking a membrane and thereby avoiding high background (However, go for the fat-free, dry variant, not the fresh one). I have no idea who originally came up with the idea of blocking a membrane with dry milk, but it has got to be a pretty good story…