What is a Western Blot?

A western blot is an assay to detect the presence of a protein in an aqueous solution by separating all proteins apart from each other in an orderly fashion and then detecting individual proteins using antibodies.

This western blot guide provides an overview of applications and principles. Western blotting is a staple experiment in the toolkit of biochemists, molecular biologists, and cell biologists. At the least, western blotting reveals the presence or absence of a protein. But used creatively, this assay can provide a lot of information.

Overview of Western Blot Principles

Western blotting involves two types of gels. The first is the stacking gel, which is used to line up the proteins in each sample into neat rows next to each other. The second gel is the separating gel, which separates each protein in the mixture according to its size. Together, the two gels produce vertical columns of proteins in each sample.

Common Western Blot Applications

Detecting the Phosphorylation States of Proteins

Many proteins are post-translationally modified by kinases, which add phosphate groups to their substrates. Western blotting is great for detecting the presence of phosphorylated proteins. Phosphorylated proteins become heavier, due to the added weight of the phosphate group, so they often migrate more slowly than their un-phosphorylated forms.

Detecting Changes in Protein Levels Across Treatment Groups

Detecting the presence and absence of a protein may seem like a simplistic assay, but when each sample represents a different treatment group, western blotting can be very informative about the amount of each protein in treatment. This also applies to detecting how a certain treatment changes the post-translational modification of a protein (i.e. phosphorylation, ubiquitination, etc.).

Detecting Changes in Protein Levels Across Time Points

Western blotting is incredibly informative for determining the effect of time on a protein. For example, if each sample is a protein mixture of cells that are in different phases of the cell cycle, then western blotting will reveal how much a protein is present or absent during each phase.

Detecting Truncated Isoforms of Proteins

Many proteins are cleaved in order to be activated or have naturally occurring truncation isoforms. Each isoform may have a different level of activity, a different target protein, or represent a different cellular state. Western blotting is great for detecting the ratio of truncated to normal isoforms of a protein.

Detecting Tagged Proteins

Some proteins are engineered, through the process of molecular cloning, to contain short sequences of amino acids that serve as a tag. Common tags include the HA-tag and the Myc-tag. These tags serve as a foreign protein epitope that does not naturally occur in the biological system being studied. Thus, the tag makes the protein easy to detect compared to all other naturally occurring proteins. An antibody directed to the tag will identify the presence and amount of the tagged protein in the western blot.