Samples were mixed with a freshly prepared reaction medium (100 mmol/L bicine
, 1.66 mmol/L phenazine ethosulfate 0.83 mg/ml of BSA, 0.5 mol/L ethanol, 0.42 mmol/L of MTT, 4.17 mmol/L EDTA-Na, and 2 U alcohol dehydrogenase) in 96-well plates and incubated at 30[degrees]C for 30 min in the dark.
Alcohol dehydrogenase, NAD, ADP-ribosyl cyclase, nicotinamide, resazurin, diaphorase, bovine serum albumin (BSA), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), bicine, riboflavin 5'-mono-phosphate (FMN), phenazine ethosulfate (PES), ethylenediaminetetraacetic acid (EDTA)-Na, alcohol dehydrogenase (ADH), and 8-Br-cADPR were purchased from Sigma-Aldrich (St.
The samples were mixed with 0.83 mg/ml BSA, 100 mmol/L bicine, 4.17 mmol/L EDTA-Na, 0.50 mol/L ethanol, 0.42 mmol/L MTT, 1.66 mmol/L PES, and 2 U ADH in 96 well plates.
For measurement of fern soluble protein concentration, fresh material was homogenized in buffer containing 20 m M Mg[Cl.sub.2], 1 mM EDTA, 10 mM Na[HCO.sub.3], 5 mM dithiothreitol, and 10 g [L.sup.-1] Polyclar in 50 mM bicine
(pH 8.2), on ice.
Conditions were as follows: 1 [micro]g of total RNA was amplified in a total volume of 50 [micro]L containing 50 mmol/L bicine
, 115 mmol/L K[C.sub.2][H.sub.3][O.sub.2], 80 g/L glycerol, pH 8.2, 0.45 [micro]mol/L primers, 300 [micro]mol/L each dNTP, 2.5 mmol/L
2-Ketoisohexanoic acid sodium salt was from Nacalai Tesque (Kyoto, Japan); N,N-bis(2-hydroxyethyl)glycine (Bicine) from Dojindo Labs (Kumamoto, Japan); bilirubin from Sigma Chemical Co., and Intralipid 10% from KabiVitrum AB.
Reagent 1 (R1) for the new method contained 2-ketoisohexanoic acid 3.0 mmol/L, [beta]-NADH 0.3 mmol/L, and LED 1.5 kU/L in 100 mmol/L Bicine buffer (pH 8.75).
Fresh-frozen leaf samples (1 g), previously ground in liquid nitrogen, were ground in 5 mL of ice cold extraction buffer [100 mM Bicine
(pH 8.1), 10 mg/ml PEG (polyethylene glycol) 4000, 7.5 mM DTT (dithiothreitol), 1 mM [Na.sub.2]EDTA] with a mortar and pestle and clarified by refrigerated centrifugation.