Amino Bi Forest-N-Demethylase（AND）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 enzymes that play an important role in the metabolism of exogenous substances, especially drugs and toxins.AND asP450An important member of the enzyme system, equivalent toCYP3A4 Subtypes are closely related to the demethylation reaction of drugs.

Measurement principle:

AND Catalyze the release of formaldehyde from amino acid residues, through Nash The colorimetric method can be used to determine the formaldehyde content and calculate itout AND Activity.

Aminobilin-N-demethylase activity assay kit P450

composition

Reagent 1: Powder×1 Bottle,4℃preservation. Add before use 100ml Dissolve distilled water thoroughly.

Reagent 3: Powder×1 Pipe,4℃Store away from light. Add before use 1ml Anhydrous ethanol, fully dissolved. Reagent 4: Powder×1Pipe,4℃preservation. Add before use 0.5mlDistilled water, fully dissolved.

Reagent 5: Powder×1 Bottle, stored at room temperature. Add distilled water before use 4ml Fully dissolved.

Standard liquid: Liquid×1 Bottle,-20℃preservation. Take before use 1.5ml EP Tube, join in 10μl Standard solution, add 990μl Distilled water, mix well to obtain0.05 mmol/L Standard formaldehyde solution,4℃preservation.

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

Self provided instruments and supplies:

Ordinary centrifuge, high-speed centrifuge, water bath, adjustable pipette, UV spectrophotometer/ELISA reader, trace quartz colorimetric dish/96 KongPlate, distilled water, anhydrous ethanol, and ice.

Crude enzyme extract：

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

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

3Remove impurities such as hemoglobin: proceed to step 2 Add to the sediment 1ml Reagent 1, tightly cover and shake thoroughly to dissolve,100 000g centrifugation 30minAbandoningThe supernatant.

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

Used on the same day.

AND Activity assay procedure:

1. spectrophotometer/Preheating of ELISA reader 30 minAdjust the wavelength to 412 nmAdjust distilled water to zero.

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

3. Care for:取 1 branchEP Tube, join in 10μl Crude enzyme solution,170μl Reagent 2,10μl Reagent 3,10μl Distilled water, mix well and place in 37℃

Water bath insulation 30minJoin now 35μl Reagent 5, mix well and place in an ice bath 5min；取outJoin later 35μl Reagent 6, mix well at room temperaturestanding 5minRoom temperature 8000rpm centrifugation 5minTake a new oneEP Tube, join in 100μl The supernatant,100μl Reagent 7, after mixing well 60℃water bath 10minThen take itoutCool with cold water 5min, in 412nmMeasure light absorption, denoted asATake care of it.

4. Measurement tube:取 1 branchEP Tube, join in 10μl Crude enzyme solution,170μl Reagent 2,10μl Reagent 3,10μl Reagent 4, mix well and place in 37℃

Water bath insulation 30minJoin now 35μl Reagent 5, mix well and place in an ice bath 5min；取outJoin later 35μl Reagent 6, mix well at room temperaturestanding 5minRoom temperature 8000rpm centrifugation 5min；取 1 Zhi XinEP Tube, join in 100μl The supernatant,100μl Reagent 7, after mixing well 60℃waterbath 10minThen take itoutCool with cold water 5min, in 412nmMeasure light absorption, denoted asAMeasurement tube.

5. Standard tube:取 1 branchEP Tube, join in 100μl Standard products,100μl Reagent 7, after mixing well 60℃water bath 10minThen take itoutUsing coldwater cooling5min, in412nm Measure light absorption, denoted asA Standard tube.

Attention: Each sample requires proper care.

AND Activity calculation:

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℃Per minute per milligram of protein catalyzed production 1nmol Formaldehyde is 1 One enzyme activity unit.

AND 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

=45×（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 of tissue per minute per gram 1nmol Formaldehyde is 1 One enzyme activity unit.

AND 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÷(V sample

÷V Sample General×W)÷T

=45×（A Measurement tube -A To take care of)÷A Standard tube÷W.

C Standard product:0.05 mmol/L=50μmol/L；V Standard product:500μl=0.0005 L; Dilution ratio:V Anti General÷V supernatant=（50+850

+50+50+175+175）÷500=2.7；CprCrude enzyme solution protein concentration（mg/ml）, requires additional testing, it is recommended to use our company BCAProtein content determination kit;VSample: Add the volume of crude enzyme solution,50μl=0.05ml；VSample total: volume of extraction solution,0.5ml；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℃Per minute per milligram of protein catalyzed production 1nmol Formaldehyde is 1 One enzyme activity unit.

AND 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

=45×（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 of tissue per minute per gram 1nmol Formaldehyde is 1 One enzyme activity unit.

AND 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÷(V sample÷VSample General×W)÷T

=45×（A Measurement tube -A To take care of)÷A Standard tube÷W.

C Standard product:0.05 mmol/L=50μmol/L；V Standard product:500μl=0.0005 L; Dilution ratio:V Anti General÷V supernatant=（50+850

+50+50+175+175）÷500=2.7；CprCrude enzyme solution protein concentration（mg/ml）, requires additional testing, it is recommended to use our company BCAProtein content determination kit;V Sample: Add the volume of crude enzyme solution,50μl=0.05ml；V Sample total: volume of extraction solution,0.5 ml；TCatalytic reaction time（min），30min.

Notes:

1The crude enzyme solution needs to be measured on the same day. If it needs to be stored, the extraction step from the crude enzyme solution should be carried out 3 Add to the sediment 0.5ml 20%After packaging glycerol,

-80℃preservation;

2Reagents three and four need to be prepared before use. If they are not used up on the same day,4℃Store away from light, usable 1 week

3The crude enzyme solution can be directly used for protein concentration determination. It is recommended to use it BCA Measure protein content using the method.

Aminobilin-N-demethylase activity assay kit P450