Typhoon rgb biomolecular imager

The dsDNA containing a 5′-conjugated FAM was prepared by annealing complementary oligonucleotides (10 μM each, Supplementary Table S1) in 20 mM Tris pH 8.0 by heating the reaction to 95°C for 10 min and allowing it to cool to room temperature slowly. For Zn-dependent EMSA reactions, 1 ng of labelled dsDNA and 100 nM purified apo-ScZur or mutants were mixed in 10 μl binding buffer (20 mM Tris–HCl (pH 8.0), 100 mM KCl, 2 mM DTT, 0.1 mg/ml BSA, 5% glycerol, 0.1 mg of poly(dI-dC) and 0–40 μM ZnSO₄) and incubated at room temperature for 20 min. For Zur-dependent EMSA reactions, 0–800 nM purified ScZur or mutants and 1 ng of labelled dsDNA were mixed in 10 μl binding buffer (20 mM Tris–HCl (pH 8.0), 100 mM KCl, 2 mM DTT, 0.1 mg/ml BSA, 5% glycerol, 0.1 mg of poly(dI-dC) and 20 μM ZnSO₄) and incubated at room temperature for 20 min. The binding mixture was then subjected to electrophoresis at 4°C on a 5% polyacrylamide gel at 130 V in TB (89 mM Trizma base, 89 mM boric acid) buffer, followed by a visualization using Amersham Typhoon RGB Biomolecular Imager with Cy2 filters. Signals from all shifted bands were combined to obtain the bound fraction, and the signal from non-shifted band was used for non-bound fraction. Data from three independent experiments were fitted to a Hill equation to obtain the n_h and the K_D-ave.