Infinite f200 fluorescence plate reader

Expression of the rMoPrP and aggregation of rMoPrP were carried out as described
previously8 (link)9 (link). In brief, a solution of rMoPrP
(2.0 μM) in NaCl/HEPES buffer (50 mM
HEPES/KOH, 300 mMNaCl, pH 7.5) was added to a 96-well plate to
create a final volume of 200 μL. The plate was incubated
at 37 °C for 72 h in a shaker-equipped plate
reader (Infinite F200 fluorescence plate reader; Tecan, Männedorf,
Switzerland) with repeated 30 s of shaking and 30 s of
pause. To determine the conversion of rMoPrP to β-sheet rich
rMoPrP aggregates, freshly prepared rMoPrP aggregates were co-incubated with
10 μM of thioflavin-T (ThT) at room temperature for
10 min. The increase in fluorescence intensity was measured using a
plate reader at an excitation and emission wavelength of 440 and
485 nm, respectively.

MutS (total concentration per monomer 0–400 nM) was mixed with DNA duplexes V–X (20 nM) in 100 µl of 25 mM Tris-HCl buffer (pH 7.5) containing 125 mM KCl, 5 mM MgCl₂, and 1 mM ADP and kept for 5 min at 20°C. Fluorescence intensity (I) and anisotropy (r) were measured using Tecan Infinite F200 fluorescence plate reader (Switzerland). Three filter sets were used, namely Green (λ_ex = 475 nm, λ_em = 525 nm), Red (λ_ex = 575 nm, λ_em = 625 nm), and FRET (λ_ex = 475 nm, λ_em = 625 nm) [35] (link). The anisotropy values for the maximal binding of MutS with the duplexes V–X were estimated (Figure S5 in File S1). Change in the efficiency of energy transfer was calculated. Each experiment was performed in 3 timed, and the SD was determined (Table 3).