Immunoblot Assay: The Western Blot


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(a) A diagram showing the process of a western blot. Step 1 – a gel is on top of a nitrocellulose membrane and filter paper is on either side. This is all sandwiched between positive and negative plates. If the proteins are hydrophobic use PVDF membrane instead of nitrocellulose. This causes the proteins to bind to the membrane. At this point there are many protein bands.  Then antibodies specific to the protein on interest are added. They bind to one of the protein bands on the membrane. Next labeled antibodies bidn to the first set of antibodies. This results in a single visible band. (b) A photo of the results shows dark bands on a white membrane.
(a) This diagram summarizes the process of western blotting. Antibodies are used to identify specific bands on the protein gel. (b) A western blot test for antibodies against HIV. The top strip is the negative control; the next strip is the positive control. The bottom two strips are patient serum samples containing antibodies. (credit a: modification of work by “Bensaccount”/Wikimedia Commons)

Immunoblot Assay: The Western Blot (OpenStax Microbiology)

After performing protein gel electrophoresis, specific proteins can be identified in the gel using antibodies. This technique is known as the western blot. Following separation of proteins by PAGE, the protein antigens in the gel are transferred to and immobilized on a nitrocellulose membrane. This membrane can then be exposed to a primary antibody produced to specifically bind to the protein of interest. A second antibody equipped with a molecular beacon will then bind to the first. These secondary antibodies are coupled to another molecule such as an enzyme or a fluorophore (a molecule that fluoresces when excited by light). When using antibodies coupled to enzymes, a chromogenic substrate for the enzyme is added. This substrate is usually colorless but will develop color in the presence of the antibody. The fluorescence or substrate coloring identifies the location of the specific protein in the membrane to which the antibodies are bound.

Typically, polyclonal antibodies are used for western blot assays. They are more sensitive than mAbs because of their ability to bind to various epitopes of the primary antigen, and the signal from polyclonal antibodies is typically stronger than that from mAbs. Monoclonal antibodies can also be used; however, they are much more expensive to produce and are less sensitive, since they are only able to recognize one specific epitope.

Several variations of the western blot are useful in research. In a southwestern blot, proteins are separated by SDS-PAGE, blotted onto a nitrocellulose membrane, allowed to renature, and then probed with a fluorescently or radioactively labeled DNA probe; the purpose of the southwestern is to identify specific DNA-protein interactions. Far-western blots are carried out to determine protein-protein interactions between immobilized proteins (separated by SDS-PAGE, blotted onto a nitrocellulose membrane, and allowed to renature) and non-antibody protein probes. The bound non-antibody proteins that interact with the immobilized proteins in a far-western blot may be detected by radiolabeling, fluorescence, or the use of an antibody with an enzymatic molecular beacon.


Parker, N., Schneegurt, M., Thi Tu, A.-H., Forster, B. M., & Lister, P. (n.d.). Microbiology. Houston, Texas: OpenStax. Access for free at:



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