Upon completion, the gel was incubated in an equilibrium buffer [25 mM Tris-HCl, 5 mM MgCl2 (pH 7.4)] for 15 min. Enzymatic activity of LDH was visualized with the aid of formazan precipitation.
Reactions were carried out at 37 °C for 25 min. Enzymatic activity was measured using a Spectramax Gemini fluorimeter (Molecular Devices, Sunnyvale, CA, USA).
INS-1E cells were harvested and incubated with isosmotic medium [ 16] containing 0.2% triton X-100 at 30°C for 2 min. Enzymatic activity of COX was measured spectrophotometrically at 550 nm based on previous reports [ 16, 17].
In the former, the shape of amplification curve showed two phases; linear amplification phase up to 90 min and plateau phase near 240 min, because the enzymatic activity of NV3Dpol is limited by about 120 min, as indicated in previous reports [ 18].
The enzyme activity was expressed in U/g dry cell weight (DCW), and one unit (U) was defined as the 1 g/L reducing sugars released per min. The enzymatic activity of bglB was determined by using p-nitrophenyl-β-d-glucopyranoside (pNPG) as the substrate.
After a further 20 min of incubation, enzymatic activity was terminated by adding 1 mL SD/mL HBSS, the suspension was triturated using a syringe and needle, and the cells collected by centrifugation as for the embryonic spinal cords.
Finally, the [Ca2+]o-dependence of ADP-evoked aggregation responses in plasma was abolished when platelets were resuspended in autologous heat-treated plasma (60°C, 30 min) to destroy enzymatic activity (Fig 2C, D; 41·1 ± 1·1 and 41·5 ± 1·9% in millimolar versus micromolar [Ca2+]o respectively).
Reactions were performed for 90 min at 37 °C, and enzymatic activity was measured on a spectrofluorometer (400 nm excitation, 505 nm emission).
To study effects of organic solvent on A. japonicus LAB01 lipase, the enzyme was incubated with different solvents at 30°C for 90 min, and the residual enzymatic activity was determined at assay conditions.
The sample including 0.9 mL homogenate and 0.1 mL of reaction buffer was heated to 37°C in a water bath for 15 min, and then the enzymatic activity was determined by measuring the changes in absorbance at 460 nm using a 96-well plate reader and expressed as U/g weight.
