Mouse anti glyceraldehyde 3 phosphate dehydrogenase monoclonal antibody

We reduced the cell extracts and liver homogenate samples with mercaptoethanol at 95 °C for 10 min. We loaded the samples at 10 μg of protein per lane and then separated them on a 12% polyacrylamide gel. Following the electrophoresis, we blotted the proteins on a nitrocellulose membrane (ClearTrans Nitrocellulose Membrane; Fujifilm Wako Pure Chemical) in a semidry blotting system (NA‐1512S; Nihon Eido, Tokyo, Japan) [20 (link)]. The nitrocellulose membranes were blocked with 2% skim milk. We then incubated the membranes with rabbit anti‐CA3 antibody (1 : 8000; Proteintech, Rosemont, IL, USA), rabbit anti‐catalase antibody (1 : 10,000; produced by our laboratory), mouse anti‐β actin monoclonal antibody (1 : 10,000; Fujifilm Wako Pure Chemical), or mouse anti‐glyceraldehyde‐3‐phosphate dehydrogenase monoclonal antibody (1 : 5000; Fujifilm Wako Pure Chemical). This was followed by incubation with horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit immunoglobulin G (IgG) antibody (1 : 10,000; SeraCare, Milford, MA, USA) or HRP‐conjugated goat anti‐mouse IgG antibody (1 : 2500; Biosource, Camarillo, CA, USA). Finally, we visualized the protein bands using ImmunoStar LD or ImmunoStar Zeta (both Fujifilm Wako Pure Chemical) with a LuminoGraph (Atto, Tokyo, Japan). We subjected the bands to densitometric analysis using imagej (National Institutes of Health, Bethesda, MD, USA).