Alcohol Dehydrogenase (ADH) Activity Assay Kit(Beijing Solarbio Science & Technology Co., Ltd)was used for ADH activity assay.
The cells are broken by ultrasonic wave in ice bath (Power: 300W, ultrasonic wave: 3s, interval: 7s, total time: 3 minutes). Centrifuge at 16000 ×g for 20 minutes at 4°C, take the supernatant and place it on ice for test.
Preheat spectrophotometer/microplate reader for 30 minutes, adjust wavelength to 340 nm, set zero with distilled water.
Keep the Reagent I in a 25°C water bath for more than 30 minutes.
a)Blank tube: Add 20 μL of distilled water, 160 μL of Reagent I and 20 μL of Reagent III to the micro quartz cuvette/ 96 well flat-bottom plate (UV plate) in turn. Mix them quickly and measure the change of absorption value at 340 nm, record the absorption value at 15 s and 75 s respectively, record them as A1 and A2. ΔAB = A1-A2. Only one or two blank tubes need to be test.
b)Test tube: Add 20 μL of supernatant, 160 μL of Reagent I and 20 μL of Reagent III to the micro quartz cuvette/ 96 well flat-bottom plate (UV plate) in turn. Mix them quickly and measure the change of absorption value at 340 nm, record the absorption value at 15 s and 75 s respectively, record them as A3 and A4. ΔAT = A3-A4.