The assay mixture containing 5 μl of revC14 or purified caspase-14 from cornified cells in 40 μl of the assay buffer was preincubated for 15 min at room temperature, and 5 μl of ICAD (0.1 mg/ml) (inhibitor of caspase-activated DNase) was added.
In a 96-microtitre plate, a reaction mixture containing extracts, 20 μL of 1 mM p-nitrophenyl α-D-glucopyranoside as a substrate and 1 unit/mL glucosidase enzyme, in 50 μL of 0.1 M sodium phosphate buffer was preincubated for 30 min at 37°C.
Briefly, 2 μL of the supernatant was added to the mixture containing 5 μL of 40 m m MG, 5 μL of 40 m m GSH and 90 μL of 50 m m sodium phosphate buffer, pH 6.6, which was preincubated at 37 °C.
LrXynA was incubated in 50 mM citrate phosphate (3 7) or phosphates (6 8) buffer and incubated at 50 °C for 10 min; for pH stability, LrXynA was preincubated at 50 °C for 3 h in the same buffers.
The nitrocellulose membrane was preincubated at room temperature in blocking buffer (0.5% milk powder, 0.5% BSA in TBS-T (10 m M Tris HCl, pH 8.0, 0.15 M NaCl, 0.05% Tween-20)) to prevent aspecific antibody binding.
The ZIKV helicase172 617 was preincubated at a concentration of 20 nmol/L in 20 μL assay buffer (40 mmol/L Tris, 80 mmol/L NaCl, 8 mmol/L MgAc2, 1 mmol/L EDTA, pH 7.5) in a 96-well plate.
Briefly, recombinant GLK protein (0.6 mM) was added to the reaction buffer (including 0.1 M Tris-HCl, pH 8.0, 15 mM ATP, 20 mM glucose and 6 mM Mg2+) which was preincubated at 75°C for 5 min.
Recombinant GLK protein (0.6 mM) was added to the reaction buffer (including 0.1 M Tris-HCl, pH 8.0, 15 mM ATP, 20 mM specific sugar, and 6 mM Mg2+) which was preincubated at 75°C for 5 min.
This solution was preincubated at 25°C for 10 min, after which 250 μL of 1% starch solution in 0.02 M sodium phosphate buffer (pH 6.9) was added at timed intervals and then further incubated at 25°C for 10 min.
