The secondary antibody is linked to a reporter enzyme that produces light or color so it can be easily imaged or detected. The researcher then incubates the membrane again, this time with a secondary antibody that recognizes and binds to the primary antibody. After incubation, the researcher washes off any unbound primary antibody. Detecting Proteins of Interestįrom here, the researcher applies a blocking treatment, which prevents non-specific antibody binding, and stains the membrane with primary antibodies that are specific to the protein of interest.

This is the step that gives the western blot process its name. The membrane carries all the protein bands originally in the gel and makes these proteins accessible to antibody detection. This process involves using an electric current to pull the proteins from the gel onto the membrane without disrupting the organization that they had in the gel. Then, the researcher transfers the proteins to a solid support known as a blotting membrane, usually through a process termed electroblotting.

This detergent makes the proteins unfold into linear chains and gives them a negative charge. The most common treatment, SDS-PAGE, utilizes polyacrylamide gels and buffers loaded with sodium dodecyl sulfate. The nature of the gel and the treatment of the sample dictate how they separate the proteins. Researchers can separate the proteins by a variety of factors, such as electric charge, molecular weight, and isoelectric point. How Western Blotting Works Separating the Proteinsįirst, the researcher separates the proteins in the sample using a method known as gel electrophoresis. Here, BioTechniques gives an overview of how western blotting works, the history behind the technique, and the additional techniques that have grown from the western blot. Researchers may also apply this technique to determine the size, quantity, molecular weight, and post-translational modifications of proteins. Western blotting, also known as immunoblotting, is a biochemical laboratory method that researchers use to detect specific protein molecules in complex protein mixtures.

Eastern blot