Use of Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) as Internal Control in Western-Blot Experiments?

Abstract

For many years glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was identified as a simple glycolytic enzyme. Particularly, GAPDH catalyzes the coinciding phosphorylation and oxidation of glyceraldehyde-3-phosphate to 1,3-biphosphoglycerate.1 Loading controls, often called “housekeeping genes”, are known to be structural and definitely expressed at high levels in most of tissues and cells. They are used to normalize the level of protein detected by confirm that protein loading is the same across the gel. Discovering and verification of many proteins is often done using Western blotting by normalizing against “housekeeping proteins”, such as GAPDH, β-actin, or β-tubulin, to emend for protein loading and factors, such as transfer efficiency.2 Recent studies, show that, besides its function in altering glyceraldehyde3-phosphate to 1, 3-biphosphoglycerate during glycolysis, GAPDH is involved in several non-metabolic processes, including transcriptional activation and cell apoptosis.3 Activated Akt2 may increase ovarian cancer cell survival via prevention of GAPDH-induced apoptosis. This effect of Akt2 is partially mediated by its phosphorylation of GAPDH at Thr-237, which results in the prevention of GAPDH nuclear translocation.3 This study prove that GAPDH is S-nitrosylated by Nitric Oxide (NO) in response to cell stress, which result in, it to bind the protein Siah1, anubiquitin ligase. The complex moves into the nucleus where Siah1 targets nuclear proteins for degradation, thus beginning controlled cell shutdown;