Anti mouse igg hrp

Cells were harvested by mechanical scraping on ice and lysed in M-PER (Thermo Scientific) in the presence of protease and phosphatase inhibitors (Sigma–Aldrich). Protein lysates were quantified using the BCA Assay (Thermo Scientific), separated with sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) using the Mini-PROTEAN Tetra Cell system (Bio-Rad) and transferred to PVDF membranes (Bio-Rad). Primary antibodies against endogenous HNF1A protein (Santa Cruz, sc-6548; 1:500), or actin (Sigma–Aldrich, A5441; 1:10000) were used, followed by HRP-conjugated secondary antibodies anti-goat IgG-HRP (Santa Cruz, sc-2354, 1:5000) and anti-mouse IgG-HRP (Bethyl labs, A90-516P, 1:10000). Chemiluminescent signals were detected using Super Signal West Dura Extended Duration substrate (Thermo Scientific).

A mouse IgG ELISA Quantitation Kit (Bethyl Laboratories, TX, USA) was used according to the manufacturer’s instructions. In brief, the immobilized antibody (goat-poly, anti-mouse IgG, Bethyl Laboratories) 10 µg/mL diluted with a carbonate buffer (0.05 M, pH 9.6) was added to a 96-well ELISA plate at 100 µL/well and incubated at 25 °C for 1 h. It was then washed five times with ELISA buffer (50 mM Tris, 140 mM NaCl, 0.05% Tween20, pH 8.0) and blocked with 1% bovine serum albumin for 1 h. Next, 100 µL of the target antibody (mouse, anti-goat IgG, #105-3102, Rockland Immunochemicals, PA, USA) was diluted with Blocking One (Nacalai Tesque, Kyoto, Japan) and added after washing five times with ELISA buffer for 1 h. A concentration of 0.0273 to 3.50 µg/mL was used as the standard solution for the calibration curve. After washing five times, 100 µL of labeled antibody (goat-poly, anti-mouse IgG-HRP, Bethyl Laboratories) 10 ng/mL diluted with ELISA buffer was added for 1 h. After washing five times, the samples were developed using the ELISA POD Substrate TMB kit (Nacalai Tesque, Kyoto, Japan) at 25 °C for 30 min. Subsequently, quenching with sulfuric acid (0.18 M), the absorbance was measured at 450 nm.