Welcome back, dear readers! Last time, we finished Day 1 of the Western blot. This issue, we’ll get into Day 2, which will complete the experiment.

After we retrieve our membrane from incubation with the primary antibody, we need to wash it and treat it with the secondary antibody. Then we can image the blot!

The secondary antibody is covalently bonded to an enzyme. Horseradish peroxidase (HRP) is one of the enzymes used for secondary antibodies. Specific to the primary antibody, the secondary antibody will only bind to the area where the protein of interest is located. To understand why, here’s a quick explanation regarding the structure of antibodies.

Think of the antibody as in the shape of a “Y.” The top branches of the Y are responsible for binding to the antigen. In our case, the antigen is our protein of interest. The “tail” end of the Y is sticking up. This allows for us to bind the secondary antibody to the primary antibody. The portion that binds to the antigen is called the variable region. It is unique in that every antigen has its own corresponding antibody.

The other end of the antibody—the one that “sticks up”—is known as the constant region. The constant region is not the same in every species: the constant region of an antibody from a rabbit will differ from the constant region of an antibody from a mouse. So if our primary antibody against the protein of interest was established in a mouse, we can then generate a secondary antibody from a rabbit that targets the constant region of the mouse’s primary antibody.

Once the secondary antibody is added, it must be left to incubate for some time. Just as with the primary antibody, the incubation ensures that the antibody is able to properly conjugate with its antigen – in this case, the constant region of the primary antibody. Next we add the chemiluminescent substrate. The substrate will interact with the HRP enzyme conjugated to the secondary antibody, and the chemical interaction will release a light signal. This enables us to detect the location of the protein of interest on the membrane.

The next step is imaging. The imaging method we will be focusing on is quite similar to the development of photographic negatives in a darkroom. The membrane is placed into a cassette. An X-ray film is placed over top of the membrane. As X-ray films are sensitive to light, it is crucial to do this step in the dark. We then leave the film to absorb the chemiluminescent reaction.

After this incubation is complete, we take the film and dunk it in the film developer, rinse it, dunk in the fixative, rinse again, and set to dry. We then observe the image. If all has worked nicely, you will see a dark band indicating the presence of your protein.