Hrp conjugated anti ha tag antibody ab1265

ELISA 96-well plates (Costar, Corning, NY, USA) were coated with 50 μL per well of cardiolipin (Sigma, St. Louis, MO, USA) prepared at 200 μg/mL in ethanol and blocked for 2 h with 4% BSA in a 20 mM Tris, 100 mM NaCl buffer at pH 7.4. To generate a binding curve, increasing concentrations of HA-DV were applied to wells. The binding data were fit to a one-site model using the Gnuplot 5.0 program (http://www.gnuplot.info/). cardiolipin binding by HA-DV variants was compared to cardiolipin binding by HA-DV at protein concentrations of 500 nM and 1000 nM. Bound HA-tagged proteins were detected with HRP-conjugated anti-HA-tag antibody (ab1265, Abcam, Cambridge, MA, USA) using a TMB (3,3′,5,5′-tetramethylbenzidine) substrate. Absorbances at 450 nm were measured on a Spectramax 340PC Microplate Reader (Molecular Devices Inc., Sunnyvale, CA, USA).