384 well low volume black round bottom polystyrene nbs microplates

Nicotinamide 1,N⁶-ethenoadenine dinucleotide (ε-NAD, Sigma-Aldrich) is a fluorescent analog of NAD⁺ and was utilized in kinetic assays as a TIR-1 substrate. TIR-1 cleaves the nicotinamide moiety from ε-NAD to release nicotinamide and etheno-ADPR (ε-ADPR), which fluoresces (λ_ex = 330 nm, λ_em = 405 nm). Enzymatic activity was assayed in Assay Buffer (50 mM Tris pH 8.0, 150 mM NaCl; final concentration) using Corning 96–well Half Area Black Flat Bottom Polystyrene NBS Microplates for a final reaction volume of 60 μL, or Corning 384-well Low Volume Black Round Bottom Polystyrene NBS Microplates for a final volume of 20 µL; reactions were initiated by the addition of ε-NAD. ε-ADPR fluorescence intensity readings were taken in real time every 15 s for 15–30 min using Wallac EnVision Manager Software and a PerkinElmer EnVision 2,104 Multilabel Reader. Fluorescence intensity readings (λ_ex = 330 nm, λ_em = 405 nm) were converted to [ε-ADPR] with an ε-ADPR standard curve, which was produced by incubating fixed concentrations (0–400 µM) of ε-NAD with excess ADP-ribosyl cyclase and plotting the peak fluorescence intensity values against [ε-ADPR]. The activity was linear with respect to time under all conditions tested.

Fluorescence polarization measurements were performed with a Safire two instrument (Tecan) at 30°C using Corning 384 Well Low Volume Black Round Bottom Polystyrene NBS microplates. Reaction volumes of 20 μL contained a fixed concentration (20 nM) of one of the indicated ^5-FAMCENP-T peptides or of a ^5-FAMCENP-C^1-21:MIS12C complex. These were mixed with increasing concentrations of the CENP-C^1-71, CENP-T^195-215, or MIS12C variants at the indicated concentrations in binding buffer (20 mM Tris-HCl, pH 8, 150 mM NaCl and 1 mM TCEP). The reaction mixtures were allowed to equilibrate for approximately 15 min at room temperature. Fluorescein (5-FAM) was excited with polarized light at 470 nm, and the emitted light was detected at 525 nm through both horizontal and vertical polarizers. No change in the observed signal (or the underlying observed dissociation constant) was detected after one-hour incubation on ice. Polarization values are shown as mean ± standard error of the mean for three replicates. Dissociation constant values (Kd) were obtained by fitting the fluorescence polarization data by non-linear least square method to a single-site binding model using the Origin software.

Fluorescence polarization measurements were performed with a Safire 2 instrument (Tecan) at 30°C. The reaction volume was 20 μL and the fixed concentrations (20 nM) of 5-FAM labeled CENP-C^1-21 peptide were mixed with increasing concentrations of the respective Mis12 variant (in the range of 1.28 pM-30 μM) in binding buffer (20 mM Tris-HCl, pH 8, 150 mM NaCl and 1 mM TCEP) in Corning 384 Well Low Volume Black Round Bottom Polystyrene NBS microplates. The reaction mixtures were allowed to equilibrate for approximately 15 min at room temperature. Fluorescein (5-FAM) was exited with polarized light at 470 nm, and the emitted light was detected at 525 nm through both horizontal and vertical polarizers. No change in the observed signal (or the underlying observed dissociation constant) was detected after one-hour incubation on ice. Polarization values are shown as mean ± standard error of the mean for three replicates and are plotted as a function of the logarithm of Mis12 concentration. Dissociation constant values (Kd) were obtained by fitting the fluorescence polarization data by non-linear least square method using the Origin software.