Synergytm2 microplate reader

The oligonucleotides (IDT) FHJ13/13-1 labeled at the 5′ end with 6-carboxyfluorescein (6-FAM) and FHJ13/13-2 were mixed in annealing buffer C (Supplementary Table S1) and were heated to 373 K for 3 min and slowly cooled to room temperature for making F13/13HJ. Purified proteins were dialyzed into buffer F (25 mM Tris-HCl pH 7.4, 100 mM NaCl, 5 mM EDTA pH8.0, 1% Glycerol) at 4 °C overnight, and the concentrations were measured by UV absorption. Protein concentration was expressed as that of a dimer. The indicated amount of mutants were mixed with F13/13HJ (20 μl total) at the final DNA concentration of 10 nM, then 15 μl was loaded on each well of a 384 well plate (Corning 3821BC), and incubate at 37 °C for 20 min (13). Positive reference samples contained all components except the enzyme. Binding was monitored through measuring change of Fluorescence Polarization (FP) using BioTek Synergy^TM2 Microplate reader. Data were analyzed by Origin software and fit to the formula: y = 1/(1 + (K/(x − 0.5*(A + x + K − ((A + x + K)^2–4 * A * x)^0.5)))) (23).
A: DNA concentration (10 nM); x: protein concentration. y: bound fraction; K: dissociation constant (Kd).

Calcium content was determined colorimetrically by the o-cresolphthalein complexone method using a QuantiChrom^TM calcium assay kit (cat. no. DICA-500; BioAssay Systems) in 0.6 M HCl extracts from cultured cells overnight at 4 °C. Briefly, 5 μl of sample was transferred to a 96-well plate. Working reagent (200 μl) was added, and the absorbance was then measured at 612 nm using a Synergy^TM 2 microplate reader from BioTek (Winooski). After decalcification, the cells were washed three times with PBS and lysed with lysis buffer. The protein content was measured with a Pierce^TM BCA protein assay kit (cat. no. 23225; Thermo Fisher Scientific). The calcium content of AVICs was then normalized to the protein content.

Antioxidant capacity of V. opulus fruit samples was determined by ABTS radical cation (ABTS), oxygen radical (ORAC) scavenging capacity, and as ferric reducing power (FRAP). Antioxidant capacity was expressed as mM Trolox equivalents (TE) per g of CE and PE extracts, and PEAF and PEWF fractions. ABTS and FRAP assays procedures have been detailed in our previous works [41 (link),46 (link)]. The ABTS^•+ monocation was generated by oxidation of ABTS with potassium persulphate. The tested sample (20 µL) was added to the ABTS^•+ solution (1 mL) and after 6 min, the absorbance at 734 nm was measured. In the FRAP assay, 90 µL of tested sample was mixed with 0.27 mL of water and freshly prepared FRAP reagent (2.7 mL). After 10 min the absorbance at 593 nm was measured. ORAC assay was carried out using a Synergy^TM2 microplate reader (BioTek Instruments Inc., Winooski, VT, USA). Peroxyl radicals were generated by the spontaneous decomposition of AAPH at 37 °C. The loss of fluorescence of fluorescein was an indication of the extent of damage from its reaction with the peroxyl radicals. Fluorescence was read at 485 nm excitation and 520 nm emission every 2 min for 2 h.