Validation of a yeast malate dehydrogenase 2 (Mdh2) antibody tested for use in western blots

Abstract

Malate dehydrogenases (Mdhs) reversibly convert malate to oxaloacetate and serve as important enzymes in several metabolic pathways. In the yeast Saccharomyces cerevisiae there are three Mdh isozymes, localized to different compartments in the cell. In order to identify specifically the Mdh2 isozyme, GenScript USA produced three different antibodies that we further tested by western blot. All three antibodies recognized the S. cerevisiae Mdh2 with different background and specificity properties. One of the antibodies had a relatively low background and high specificity and thus can be used for specific identification of Mdh2 in various experimental settings.

A piggybacking mechanism enables peroxisomal localization of the glyoxylate cycle enzyme Mdh2 in yeast

“…To ensure that the peroxisomal localization of Mdh2, and its dependence on Mdh3 and Pex5, was not a result of the GFP tagging, we used a specific antibody that recognizes Mdh2 but not the other Mdh isoforms ( Gabay-Maskit et al, 2018 ). Analysis of subcellular fractions clearly demonstrated that native and untagged Mdh2 also co-sediments with peroxisomes ( Fig.…”

“…and anti-Por1 (1:10,000; Kerssen et al, 2006 ). The Mdh2 antibody was produced by GeneScript, USA, using a specific Mdh2 peptide as an immunogen (MPHSVTPSIEQDSLC), and was previously verified by us for specificity and activity (1:2500; Gabay-Maskit et al, 2018 ). The immunodecorated membranes were incubated with IRDye 800CW goat anti-rabbit IgG or IRDye 800CW goat anti-mouse IgG (LI-COR Bioscience) as secondary antibodies, and detected with an Odyssey Infrared imaging system (LI-COR Bioscience).…”