Thus, using a reaction buffer that has an effective low Mg2+ concentration, GTPase and GAP-assisted GTPase can be performed without the need for GEF proteins.
Firefly luciferase activity was determined in a 96-well plate reader (Victor3V; PerkinElmer) as described previously [ 17] using a reaction buffer containing 20 mM tricine, 2.67 mM MgSO4, 0.1 mM EDTA, 33.3 mM DTT (dithiothreitol), 270 μM co-enzyme A, 470 μM D-luciferin and 530 μM ATP at pH 7.8 [ 18].
To detect levels of individual ROS generated by PSII-driven electron transport, we used a reaction buffer containing 0.1 M sucrose, 10 mM NaCl, 10 mM KCl, 5 mM MgCl2, 10 mM Tricine, 1 mM KH2PO4, and 0.2% BSA (pH 8.0).
Each reaction was done in triplicate using a reaction solution containing SYBR Green buffer and the primers (0.3 mmol/l, Supplementary Table 3).
PCR was performed using a reaction mixture containing 1× PCR buffer; 0.2 mM (each) dATP, dTTP, dGTP, and dCTP; 0.2 mM of each primer; 2 U of EcoTaq DNA polymerase (Ecogen, TaKaRa Biotechnology, Japan), and ultrapure water to a final volume of 50 μl.
All the assays were prepared as master mixes immediately before use in a reaction buffer containing 20 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl2, and 2 mM TCEP at pH 7.5.
COX activity was measured by detecting the decrease in absorbance at 550 nm in a 96-well plate at 30°C, using 200 µl of a reaction buffer (Potassium phosphate 100 mM, pH 7.0) containing 0.1% n-Dodecylmaltoside and 0.1 mM purified reduced cytochrome c.
The dark particles collected by centrifugation at 5000 g for 10 min were subjected to three washes using PBS followed by treatment using Reaction Buffer (10.0 mM Tris, 1.0 mM CaCl2 and 0.5 % SDS, pH 7.8) with 10 μL of 10 mg/mL Proteinase K and incubated at 37 °C.
A solution was prepared by serially diluting a LAMP reaction solution with a known concentration (28 μg DNA/25 μl) using LAMP reaction buffer in order to systematically investigate the reaction between the LAMP product and PEI.
Prior to application of the sample, the chips were blocked using a solution of 2% BSA (Bovine Serum Albumin, Fraction V, Calbiochem, Germany), and washed 2 times using kinase reaction buffer.
Assays were carried out at 25 °C by using the reaction buffer of 50 mM Na-phosphate buffer (pH 7.8) including 0.033 mM NBT, 10 mM l-methionine, 0.66 mM Na2EDTA and 0.0033 mM riboflavin.
