Spectrophotometric detection kit

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　Introduction to spectrophotometric detection kit：

PDC mainly exists in yeast and is one of the key enzymes in ethanol fermentation, catalyzing the decarboxylation of pyruvate to acetaldehyde.

Measurement principle:

PDC catalyzes the decarboxylation of pyruvate to acetaldehyde, and ethanol dehydrogenase (ADH) is added to further catalyze the reduction of acetaldehyde to ethanol and NAD+by NADH; NADH has an absorption peak at 340nm, while NAD+does not; Calculate PDC activity by measuring the rate of decrease in 340nm light absorption.

Self provided instruments and supplies:

Mortar, ice, desktop centrifuge, UV spectrophotometer, 1ml quartz colorimetric dish, adjustable pipette, and double distilled water.

Composition and preparation of the agent:

Extraction solution: 100mL of liquid in 1 bottle, stored at 4 ℃.

Reagent 1: Liquid 20mL x 1 bottle, stored at 4 ℃.

Reagent 2: Powder x 1 bottle, stored at 4 ℃ in the dark. Dissolve in 1mL of distilled water before use; Unused reagents should be packaged and stored at -20 ℃, and repeated freezing and thawing are prohibited.

Reagent 3: Powder x 1 bottle, stored at 4 ℃ in the dark. Dissolve in 1mL of distilled water before use; Unused reagents should be packaged and stored at -20 ℃, and repeated freezing and thawing are prohibited.

Reagent 4: Powder x 1 bottle, stored at 4 ℃ in the dark. Dissolve in 1mL of distilled water before use; Unused reagents should be packaged and stored at -20 ℃, and repeated freezing and thawing are prohibited.

Measurement operation table:

Enzyme activity calculation formula:

a. The calculation formula for measuring with a trace quartz colorimetric dish is as follows

(1) Calculated based on sample protein concentration:

Enzyme activity definition: Under the conditions of pH 7.2 and temperature of 37 ℃, the amount of enzyme required to catalyze the production of 1nmol H2O2 per minute per milligram of tissue protein is one enzyme activity unit (U).

DAO activity (nmol/min/mg prot)=× V anti total ÷ (V sample × Cpr) ÷ T=18 × A460 ÷ Cpr

(2) Calculated by sample quality:

Enzyme activity definition: The amount of enzyme required to catalyze the production of 1nmol H2O2 per minute per gram of tissue at pH 7.2 and temperature of 37 ℃ is defined as one unit of enzyme activity.

DAO activity (nmol/min/g fresh weight)=× V reverse total ÷ (V sample ÷ V sample total × W) ÷ T=18 × A460 ÷ W

(3) Calculated by the number of cells:

Enzyme activity definition: The amount of enzyme required to catalyze the production of 1nmol H2O2 per minute per 104 cells at pH 7.2 and temperature of 37 ℃ is defined as one unit of enzyme activity.

DAO activity (nmol/min/104cell)=× V inverse total ÷ (V like ÷ V like total × number of cells) ÷ T=18 × A460 ÷ number of cells

(4) Calculated by liquid volume

Enzyme activity definition: The amount of enzyme required to catalyze the production of 1nmol H2O2 per minute per milliliter of serum at pH 7.2 and temperature of 37 ℃ is defined as one unit of enzyme activity.

DAO activity (nmol/min/mL)=× V total ÷ V sample ÷ T=18 × A460

ε: Oxidation type ortho fennel amine millimolar extinction coefficient: 7.5 L/mmol/cm; d: Colorimetric dish diameter, 1cm; V-total: total reaction volume, 0.2mL; V-sample: sample volume in reaction, 0.05mL; V-total: volume of added extraction solution, 1mL; Cpr: sample protein concentration, mg/mL； W: Sample quality, g; T: reaction time, 30 minutes

b. The calculation formula for measuring with a 96 well plate is as follows

(1) Calculated based on sample protein concentration:

Enzyme activity definition: Under the conditions of pH 7.2 and temperature of 37 ℃, the amount of enzyme required to catalyze the production of 1nmol H2O2 per minute per milligram of tissue protein is one enzyme activity unit (U).

DAO activity (nmol/min/mg prot)=× V anti total ÷ (V sample × Cpr) ÷ T=36 × A460 ÷ Cpr

(2) Calculated by sample quality:

Enzyme activity definition: The amount of enzyme required to catalyze the production of 1nmol H2O2 per minute per gram of tissue at pH 7.2 and temperature of 37 ℃ is defined as one unit of enzyme activity.

DAO activity (nmol/min/g fresh weight)=× V reverse total ÷ (V sample ÷ V sample total × W) ÷ T=36 × A460 ÷ W

(3) Calculated by the number of cells:

Enzyme activity definition: The amount of enzyme required to catalyze the production of 1nmol H2O2 per minute per 104 cells at pH 7.2 and temperature of 37 ℃ is defined as one unit of enzyme activity.

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