μL)

Reagent 2 (μL)

Reagent 3 working solution (μL)

Reagent 4 (μL)

Reagent 5 (μL)

Reagent 6 (μL)

Total amount of mixture reagent (μL)

300 × (n+2*)

[Note] n refers to the number of sample.

2*: Prepare 2 more tubes of substrate working solution.

2.    Operation procedure

1)    Pretreatment

(1)  Control tube: add 50 μL of sample into 2 mL EP tube, then incubate in 100℃ water bath for 2 min.

Sample tube: add nothing.

(2)  Add 300 μL of substrate working solution (preheated for 5 min) to each tube.

(3)  Control tube: add nothing.

Sample tube: add 50 μL of sample.

(4)  Mix fully and incubate at 37℃ water bath for 20 min, then incubate in 100℃ water bath for 2 min to stop the reaction.

(5)  Add 850 μL of double distilled water to each tube.

(6)  Mix fully and centrifuge at 3100 g for 10 min, then take 750 μL of supernatant to the new corresponding 2 mL EP tube for chromogenic reaction.

2)    Chromogenic reaction

(1)  Add 15 μL of Reagent 7 to each tube.

(2)  Mix fully and stand at room temperature for 15 min. Set spectrophotometer to zero with ddH2O and measure the OD value of each tube at 324 nm with cuvettes (1 cm optical path, 2 mm internal diameter).

1)    Pretreatment

2)    Chromogenic reaction