Clariostar plus spectrophotometer

Total RNA was isolated for sequencing and real-time PCR using Total RNA Mini Plus (A&A Biotechnology, Gdańsk, Poland) according to the manufacturer’s recommendations. Total RNA concentration and purity were determined by measuring absorbance at 260/280 nm and 260/230 nm using a CLARIOstar Plus spectrophotometer (BMG LABTECH, Ortenberg, Germany).

Fluorescence of 150 nM Mag-Fura-2 (ThermoFisher Scientific) (Ex/Em 340/510 nm) saturated with Zn(II) was measured on a CLARIOstar Plus spectrophotometer (BMG Labtech) at 25°C in response to increasing concentrations of metal-free protein. Five technical replicates of each sample were analyzed in a black half-volume 384-well microtiter plate (Greiner Bio One). All experiments were performed in Chelex-100 treated 50mM MOPS (pH 7.2) 200 mM NaCl buffer. Fluorescence values were analyzed using a one-site non-linear fit model in GraphPad Prism (Version 8) using the experimentally-derived K_D of Mag-Fura-2 (47 nM) to determine the K_Dfor Zn(II) binding by AdcAII.