Epoch 2 plate reader

The activity assays were modified for the Epoch 2 plate reader (BioTek, Burlington, VT) from methods described previously [42 (link), 47 (link)]. Deamino-NADH (dNADH) was used to avoid oxidation by the alternative NADH oxidase in E. coli membranes [48 (link)]. In brief, NADH oxidase activity assays were started with 0.25 mM dNADH (extinction coefficient 6.22 mM⁻¹ cm⁻¹) and the absorbance was monitored at 340 nm for 2 minutes. dNADH oxidase activity assays were performed in 50 mM MOPS, 10 mM MgCl₂, pH 7.3 at room temperature. The rate of dNADH oxidase from membranes of BA14/pBAD33(A-N) was about 1.0 μmoles dNADH per min per mg protein. Proton translocation assays were conducted by measuring the fluorescence quenching of the acridine dye ACMA (9-amino 3- chloro 2-methoxy acridine) as a ΔpH indicator using excitation and emission wave lengths of 410 and 490 nm, respectively. The uncoupler FCCP was added to 1 μM final concentration from a 1 mM ethanol stock to eliminate the buildup of a proton gradient during NADH oxidase assays. Proton translocation assays were conducted in 50 mM MOPS, 5 mM MgCl₂, 50 mM KCl, at pH 7.3 at 20°C, with additions of 1 μM valinomycin and 1 μM ACMA. The reactions were initiated with dNADH (0.25 mM) and after about 2 min the proton gradient was collapsed by the addition of FCCP (1 μM).

Phage ELISA was performed as described previously except that the 384-well plate was used instead of the 96-well plate.^{22 (link)} Briefly, the wells of the 384-well ELISA plate (Nunc) were coated with neutravidin (Thermo Fisher) for 1 h at room temperature. The wells were washed with PBS-T (PBS containing 0.1% Tween 20) and blocked with PBS containing 0.5% (w/v) BSA (Gemini Bio) for 1 h. After removing the blocking buffer, biotinylated antigens were added to each well and washed three times with PBS-T. The 5-fold dilution of the cell culture supernatants containing phage were added to the wells and incubated for 30 min. After washing the wells with PBS-T three times, anti-M13HRP (Sino Biological) was added to the wells. SIGMAFAST™ OPD (Sigma) was used as a substrate and 2 M HCl was used as a quenching solution. The absorbance at 490 nm was measured using a BioTek Epoch 2 plate reader (BioTek).