Aniline-4-hydroxylase (AH)Activity assay kit instruction manual

Micro method 100 pipe/48 sample

Before formal testing, it is necessary to take 2-3 Make predictions based on samples with significant expected differencesMeasurement significance:

cytochromeP450 Enzymes are a group of isoenzymes mainly present in the liver, playing an important role in the metabolism of exogenous substances, especially drugs andMetabolism of toxins.AH inP450 Equivalent to in the enzyme systemCYP2E1 Subtype,CYP2E1 Not only does it participate in the metabolism of drugs, but it can also catalyze multiple reactionsThe activation process of pre cancerous and pre toxic substances.

Measurement principle:

AH Produced after catalytic hydroxylation of aniline 4-Aminophenol, further converted into phenol-Indole complex, in 630nm There is a characteristic absorption peak at this location; throughOver testing 630nmCalculate the rate of increase in absorbanceAHActivity.

Aniline-4-hydroxylase (AH) activity assay kit

composition

Reagent 1: Powder×1 Bottle,4℃preservation. Add before use 100ml Distilled water, fully dissolved. Reagent 3: Powder×1Bottle,4℃preservation. Add before use 10mlDistilled water, fully dissolved. Reagent 4: Powder×1Bottle,4℃preservation. Add before use 5mlDistilled water, fully dissolved.

Reagent 6: Powder×1 Bottle (corrosive reagent),4℃Store away from light. Add before use 10ml Distilled water, fully dissolved. Reagent 7: Powder×1Bottle,4℃preservation. Add before use 10mlDissolve distilled water thoroughly.

Standard liquid: Liquid×1 Bottle,10μmol/L，4℃Store away from light.

The specific product parameters are subject to the parameters in the product manual received

Self provided instruments and supplies:

Ordinary centrifuge, high-speed centrifuge, visible spectrophotometer/ELISA reader, micro glass colorimetric dish/96 Orifice plate, double distilled water, and ice.

Extraction of crude enzyme solution:

1、Excluding large molecular substances such as nuclei and mitochondria:About it 0.5g Organize, join 1ml Reagent 1: Grind thoroughly on ice,10 000g 4℃，centrifugation 30minTake the supernatant and transfer it to a high-speed centrifuge tube.

2、Crude microsomes: 100 000g 4℃, centrifugation 60minDiscard the supernatant.

3、Excluding impurities such as hemoglobin:Step by step 2 Add to the sediment 1ml Reagent 1, tightly cover and shake thoroughly to dissolve,100 000g centrifugation 30minAbandoning

The supernatant.

4、Final microsomes:Step by step 3 Add reagent 2 to the precipitate 0.5mlAfter sealing tightly, shake thoroughly to dissolve the crude enzyme solution, which is ready for testing.

Measurement operation：

1. spectrophotometer/Preheating of ELISA reader 30 minAdjust the wavelength to 630 nmAdjust distilled water to zero.

2. Reagent 3 is placed in 37℃Preheating in a water bath 30min.

3. Reagent 5 is placed in an ice bath for pre cooling 30min.

4. Standard tube:取 0.5 ml EP Tube, join in 100μl Standard solution,100μl Reagent 6,100μl Reagent 7, mix well and let it stand at room temperature 30min，absorb 200μlMicro glass colorimetric dish/96Orifice plate,630 nmMeasure light absorption, denoted asAStandard tube.

5. Care for:取 0.5 ml EP Tube, join in 50μl Crude enzyme solution,100μl Reagent 3,50μl Distilled water, mixed thoroughly 37℃Insulation in water bath 30min；

add in 100μl Reagent 5, mix well and take an ice bath 5min，11000rpm，4℃, centrifugation 10min；取 100μl Clear liquid, add new one 0.5 mlEP Management; add in 100μl Reagent 6,100μl Reagent 7, mix well and let it stand at room temperature 30min, absorb 200μl Micro glass colorimetric dish/96 KongBoard,630 nmMeasure light absorption, denoted as ATake care of it.

6. Measurement tube:取 0.5 ml EP Tube, join in 50μl Crude enzyme solution,100μl Reagent 3,50μl Reagent 4, after mixing well 37℃Insulation in water bath 30min；

add in 100μl Reagent 5, mix well and take an ice bath 5min，11000rpm，4℃, centrifugation 10min；取 100μl Clear liquid, add new one 0.5 mlEP Management; add in 100μl Reagent 6,100μl Reagent 7, mix well and let it stand at room temperature 30min, absorb 200μl Micro glass colorimetric dish/96 KongBoard,630 nmMeasure light absorption, denoted as AMeasurement tube.

AH Activity calculation formula：

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

（1）Calculated by protein concentration

Definition of active unit:37℃Catalytic production per minute per milligram of protein 1nmol 4-Aminophenol is 1 One enzyme activity unit.

AH activity(nmol/min/mg prot) =C reference standard×V reference standard×（A Measurement tube -A To take care of)÷A Standard tube×dilution factor÷（Cpr×VSample)÷T

=2×（A Measurement tube -A To take care of)÷A Standard tube÷Cpr.

（2）Calculated based on sample quality

Definition of active unit:37℃Catalytic production per gram of sample per minute 1nmol 4-Aminophenol is 1 One enzyme activity unit.

AHactivity(nmol/min/g fresh weight)=C reference standard×V reference standard×（A Measurement tube -A To take care of）÷A Standard tube×dilution factor÷(W×V sample)÷T

=2×（A Measurement tube -A To take care of)÷A Standard tube÷W

CStandard product:10μmol/L；VStandard product:100μl=1×10^-4L; Dilution ratio: VAnti General÷Vsupernatant=300μl÷100μl=3；Cpr: RoughEnzyme solution protein concentration（mg/ml）, requires additional testing, it is recommended to use our company BCA Protein content determination kit;W : Sample quality;VSample: Add the volume of crude enzyme solution to the reaction system, 50μl=0.05 ml； TCatalytic reaction time（min），30min.

b.use 96 The calculation formula for orifice plate measurement is as follows

（1）Calculated by protein concentration

Definition of active unit:37℃Catalytic production per minute per milligram of protein 1nmol 4-Aminophenol is 1 One enzyme activity unit.

AH activity(nmol/min/mg prot) =C reference standard×V reference standard×（A Measurement tube -A To take care of)÷A Standard tube×dilution factor÷（Cpr×VSample)÷T

=2×（A Measurement tube -A To take care of)÷A Standard tube÷Cpr

（2）Calculated based on sample quality

Definition of active unit:37℃Catalytic production per gram of sample per minute 1nmol 4-Aminophenol is 1 One enzyme activity unit.

AH activity(nmol/min/g fresh weight) =C reference standard×V reference standard×（A Measurement tube -A To take care of）÷A Standard tube×dilution factor÷(W×V sample)÷T

=2×（A Measurement tube -A To take care of)÷A Standard tube÷W

C Standard product:10μmol/L；V Standard product:100μl=1×10^-4L; Dilution ratio:V Anti General÷Vsupernatant=300μl÷100μl=3；CprCrude enzyme solution protein concentration（mg/ml），Additional testing is required, it is recommended to use our company BCA Protein content determination kit; W : Sample quality; VSample: Add the volume of crude enzyme solution to the reaction system, 50μl=0.05 ml； TCatalytic reaction time（min），30min.

Aniline-4-hydroxylase (AH) activity assay kit