Red mold su-N-demethyltransferase (ERND)Activity assay kit instruction manual

Micro method 100T/48S

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

Cytochrome P450 Enzymes are a group of enzymes mainly present in the liver, which play a crucial role in the metabolism of exogenous substances, especially drugs and toxinsIt plays an important role. ERND inP450 Equivalent to in the enzyme systemCYP2B Subtypes are closely related to demethylation in drug metabolism. CYP2B haveCatalyzing substrates to form inactive and easily excreted metabolites, which have detoxifying effects, and can also enable certain drugs to pass throughCYP2B metabolic activation.

Measurement principle:

ERNDCatalyze the release of formaldehyde from red mold su, throughNash colorimetric determination of formaldehyde content can be used to calculate ERND 活性。

Red mold su-N-demethyltransferase activity assay kit P450 series

composition

Reagent 1: Powder x 1 Bottle, 4Store at ℃. Add before use100ml Dissolve in distilled water.

Reagent 3: Powder x 1 tube, 4Store at ℃. Add before useDissolve 1ml of distilled water thoroughly.Reagent 4: Powder x 1 Bottle, 4Store at ℃. Add before use0.5ml Distilled water, fully dissolved.Reagent 5: Powder x 1 Bottle, 4Store at ℃. Before use, add distilled waterDissolve 4.5ml thoroughly.

Standard solution: Liquid x 1 Bottle, -20Store at ℃. Take before use1.5ml EP Tube, join in 10μlStandard solution, add 990 μ l distilled water, mix well to obtain 0.05 mmol/LStandard formaldehyde solution, 4℃保存。

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

Self provided instruments and supplies:

Ordinary centrifuge, high-speed centrifuge, adjustable pipette, visible spectrophotometer/enzyme-linked immunosorbent assay reader, trace quartz colorimetric dish/96 Orifice plate, distillationWater and ice.

Extraction of crude enzyme solution:

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

2Crude microsomes:100 000g，4℃, centrifugation60min， Discard the supernatant.

3Excluding impurities such as hemoglobin:Step by step 2 Add to the sediment 1mlReagent 1, tightly cover and shake thoroughly to dissolve, 100000gcentrifugation 30min， abandon

The supernatant.

4Final microsomes:Step by step 3 Add reagent 2 to the precipitate 0.5ml， Thoroughly shake and dissolve, i.e. crude enzyme solution, to be tested. The test liquid needs to be used on the same day.

Measurement operation:

1. Spectrophotometer/Preheating of ELISA reader 30 minAdjust the wavelength to 412 nm， Adjust distilled water to zero.

2. Reagent 2 is placed in 37Preheating in a ℃ water bath30 min.

3. Care for:取 0.5ml EP Tube, join in10μl Crude enzyme solution, 170 μ l Reagent 2, 10 μ l Reagent 3, 10 μ l Distilled water, mix well and place in37℃Water bath insulation 30minJoin now 35μlReagent 5, mix well and place in an ice bath 5min； 取出Join later 35 μ l of reagent six, mixed well at room temperature

standing 5minRoom temperature 8000rpmcentrifugation 5min； Get a new EPTube, join in 100μl Supernatant, 100 μ l Reagent 7, after mixing well 60 ℃ water bath10min， Then take outCool with cold water 5min, in 412nmMeasure light absorption, denoted asA takes care of it.

4. Measurement tube:取 0.5ml EP Tube, join in10μl Crude enzyme solution, 170 μ l Reagent 2, 10 μ l Reagent 3, 10 μ l Reagent 4, mix well and place in37℃

Water bath insulation 30minJoin now 35μlReagent 5, mix well and place in an ice bath5min； 取出Join later 35 μ l of reagent six, mixed well at room temperaturestanding 5minRoom temperature 8000rpmcentrifugation 5minGet new EPTube, join in 100 μ l supernatant，100μl Reagent 7, after mixing well 60℃ water bath10min， Then take outCool with cold water 5min, in 412nmMeasure light absorption, denoted asA measuring tube.

5. Standard tube:取 0.5ml EPTube, join in 100μlStandard product, 100 μ l Reagent 7, after mixing well 60℃ water bath10min， Then take it out,Using cold

water cooling 5min, in 412nm Measure light absorption, denoted asA Standard tube.

Attention: Each sample requires proper care.

ERND Activity calculation formula:

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

(1).Calculated based on protein concentration:

Definition of active unit: 37At ℃, 1nmol of protein is catalyzed per milligram per minuteFormaldehyde is 1 enzyme activity unit.

ERND Activity (nmol/min/mg)prot) = C Standard product x V reference standard×（A Measurement tube A To take care of）÷A Standard tube x dilution factor÷（Cpr×V

sample）÷T

=45×（A Measurement tube A Care (÷ A) Standard tube ÷ Cpr.

(2).Calculated based on sample quality:

Definition of active unit: 37At ℃, 1nmol is catalyzed per gram of sample per minuteFormaldehyde is 1 enzyme activity unit.

ERND Activity (nmol/min/g) Fresh weight=C Standard product x V Standard product X (A) Measurement tube A Care (÷ A) Standard tube x dilution ratio ÷ (W x V sample) ÷ T

=45×（A Measurement tube A Care (÷ A) Standard tube÷W

CStandard: 0.05mmol/L=50μmol/L； VStandard product: 100 μ l=1 × 10^-4 L. Dilution ratio: VCounter total ÷ V Supernatant=(50+850)

+50+50+175+175）÷500=2.7； Cpr: crude enzyme solution protein concentration (mg/ml), requires additional testing, it is recommended to use our companyBCA

Protein content determination kit; W: Sample quality, g；V Sample: Add crude enzyme solution volume, 10 μ l=0.01ml; T: Catalytic reaction time（min），

30min.

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

(1).Calculated based on protein concentration:

Definition of active unit: 37At ℃, 1nmol of protein is catalyzed per milligram per minuteFormaldehyde is 1 enzyme activity unit.

ERND Activity (nmol/min/mg)prot) = C Standard product x Vreference standard×（A Measurement tube A To take care of）÷A Standard tube x dilution factor÷（Cpr×V

sample）÷T

=45×（A Measurement tube A Care (÷ A) Standard tube ÷ Cpr.

(2).Calculated based on sample quality:

Definition of active unit: 37At ℃, 1nmol is catalyzed per gram of sample per minuteFormaldehyde is 1 enzyme activity unit.

ERND Activity (nmol/min/g) Fresh weight=C Standard product x V Standard product X (A) Measurement tube A Care (÷ A) Standard tube x dilution ratio ÷ (W x V sample) ÷ T

=45×（A Measurement tube A Care (÷ A) Standard tube÷W

CStandard: 0.05mmol/L=50μmol/L； VStandard product: 100 μ l=1 × 10^-4 L. Dilution ratio: VCounter total ÷ V Supernatant=(50+850)

+50+50+175+175）÷500=2.7； Cpr: crude enzyme solution protein concentration (mg/ml), requires additional testing, it is recommended to use our company BCAProtein content determination kit; W: Sample quality, g；V Sample: Add crude enzyme solution volume, 10 μ l=0.01ml；T: Catalytic reaction time（min）， 30min。

Red mold su-N-demethyltransferase activity assay kit P450 series