Western Blot

By Christina Towers, PhD.

By Christina Towers, PhD.

By Christina Towers, PhD.

By Christina Towers, PhD.

By Jamshed Arslan Pharm.D.

The Western Blot – a tried and true experimental protocol where protein structures are separated via molecular weight/charge and transferred to a membrane before visualization by a chemiluminescent solution (say that three times fast!). Seems simple, right?  While the step-by-step process of a western blot has for the most part remained the same over the years, variations in solutions, procedures and reagents may increase the efficacy of your results.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a well-known housekeeping gene with functions in glycolysis. Many biologists are familiar with the gene and use GAPDH antibodies for a loading control when performing western blots. However, this primarily cytoplasmic protein is an essential metabolic regulator and has been shown to be involved in a variety of cellular processes like DNA repair, membrane fusion, and cell death (1).

LC3B is subunit component of the LC3 autophagy biomarker associated with microtubule-associated proteins MAP1A and MAP1B and one of the best characterized markers to date. In resting state, it is cytosolic, but upon activation, is lapidated and becomes embedded in the autophagosomal membrane.

LC3B, also known as microtubule-associated protein 1 light chain 3 beta (MAP1LC3B), is an autophagy gene that contributes appreciably to protein degradation. Autophagy is a highly synchronized and dynamic catabolic degradation activity that plays an essential role in cellular maintenance, development, antigen presentation and cell death.

Microtubule-associated protein light chain 3 (LC3) is considered one of the definitive markers of autophagy, and its use is widespread in labs throughout the world. Despite its popularity, there are several considerations when employing LC3 antibodies in immunoassays, Western blots in particular.