9 Western blot intro

Intro to western blotting:

Western blotting is an immunodetection technique used to detect and quantify the abundance of specific proteins in complex biological samples. This method of identifying proteins is based on two distinguishing features of proteins: molecular mass and antibody binding specificity. Because it is so accurate, Western blotting is the method of choice to confirm positive test results for HIV, lupus, and mad cow disease. It is used by researchers to quantify protein expression (abundance) levels, study protein interactions, and determine the post-translational modifications of proteins.

How to perform a Western blot:

First, proteins are extracted from a sample of cells or tissue. Extracted proteins are loaded into a gel and separated according to size using an electrical current. Following electrophoresis, proteins are transferred from the gel onto a paper-like membrane. Proteins, negatively charged by the SDS, migrate out of the gel and bind to the surface of a membrane creating a western, or protein blot. The proteins blotted onto the membrane form a mirror image of proteins separated in the original gel. Molecules on the surface of the membrane are now accessible for further analysis.

Why is it necessary to transfer the proteins from the gel to the nitrocellulose membrane?

First, since the proteins are contained within the gel and embedded within the polyacrylamide matrix, antibodies would have difficulty reaching the proteins. Second, the gel is fragile and can easily break during analysis while a membrane is more stable and durable.

Blocking and Probing

Once proteins are transferred to the membrane (the ‘blot’), the next step is to probe the blot with an antibody that will detect a particular protein of interest. First the blot is incubated in a protein-rich solution often derived from powdered milk protein. Incubating the blot with milk effectively coats the remaining surface area of the membrane (where no proteins have been blotted) and blocks nonspecific protein binding sites. Next, the blot is incubated with a primary antibody. The primary antibody is designed to specifically bind the protein under study. In this week’s lab, we will be using an antibody that binds to a subunit of a myosin light chain protein. We then perform washes to remove unbound antibody and ensure the primary antibody is only attached to its specific target. After these washes, primary antibody should only be attached to your protein of interest, which is bound to the membrane. The membrane is then incubated with an enzyme-linked secondary antibody that has been engineered to bind specifically to the primary antibody. The enzyme portion of secondary antibodies enable visualization of the protein of interest.

Western blot image by Biochemlife, CC-BY-SA.

Detection by a colorimetric enzyme:

In our experiment, we will use a colorless colorimetric (color-producing) enzyme substrate to visualize a myosin protein on our blots. The enzyme that is linked to the secondary antibody oxidizes the colorimetric substrate into an insoluble purple precipitate that leaves visible deposits on the membrane at the precise location of the blotted myosin protein. The combined blotting and immunodetection procedure is used to determine the exact position of the protein. The precise molecular mass of myosin can then be determined for each sample by constructing a standard curve from the prestained standards run alongside the protein samples in the gel. We can determine the relative abundance of myosin across different fish samples and determine the evolutionary conservation of this muscle protein.