Synergy 4 multi well plate reader

Each aliquot was quenched with 0.5 M EDTA and incubated in a 96-well Neutravidin coated plate (15 pmol biotin binding capacity per well, Thermo Scientific) in Tris-buffered saline (TBS, 25mM Tris-HCl and 150mM NaCl) containing 0.1% BSA and 0.05% Tween 20 for 1h. Following incubation, each well was washed with the TBS buffer and then incubated with mouse anti-phosphotyrosine monoclonal antibody 4G10 (Millipore, 1:10,000 dilution in TBS buffer) for 1h. Following incubation, each well was washed with TBS buffer and incubated with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG) secondary antibody (Abcam) (1:1000 dilution) for 1h. Wells were then washed and treated with Amplex Red reaction buffer (Amplex Red reagent, Invitrogen, 20 mM H₂O₂ and sodium phosphate buffer) for 30 min. Fluorescence was measured using a Synergy4 multiwell plate reader (Biotek) with an excitation wavelength of 532 nm and emission wavelength of 590 nm.