Lb 940 plate luminometer

Before measurement biotinylated bi-functional 2′-F-RNA aptamers (O79t1-H9t11, H9t11-L-O79t1 or O79t1-L-H9t11) were folded in DPBSM by heating at 90 °C for 5 min and cooling down for 10 min at 25 °C. Then Tween 20, BSA and E. coli tRNA were added as nonspecific competitors for final concentrations of 0.05%, 0.01%, and 0.1%, respectively. The 25 nM solution of biotinylated bi-functional 2′-F-RNA aptamers (50 μL) were added into streptavidin activated wells (10 μg mL⁻¹ in PBS, pH 7.5, 50 μL in each well, overnight at 4 °C) in binding buffer (DPBSM, 0.05% Tween 20, 0.01% BSA, 0.01% E. coli tRNA) and incubated with shaking for 30 min at room temperature.
Then, the wells were washed, and 50 μL aliquots of the mixed solution of human hemoglobin (final concentration from 100 to 1.6 nM) and His-Obe (final concentration of 100 nM) in binding buffer were added into the wells and incubated for 40 min at room temperature.
Aliquots of 100 nM His-Obe in binding buffer were placed into the control wells. After washing the wells, bioluminescence of bound obelin was initiated by injection of 0.1 M CaCl₂ in 0.1 M Tris–HCl, pH 8.8 (50 μL) and measured with Mithras LB 940 plate luminometer (Berthold, Germany).
The signal was integrated for 5 s. Signals from the control wells were subtracted from those obtained from the respective aptamer-containing wells.