Acetyl Dan alkaline esterase（AchE）Activity assay kit instruction manual

Micro method 100T/96S

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

AchE Belonging to serine acid hydrolase, it is widely present in various animal tissues and serum.AchE Catalytic Acetyl Dan Base（Ach）Hydrolysis, inPlays an important role in nerve conduction regulation.

Measurement principle:

AchEcatalysisAchHydrolysis generates dan base, which reacts with disulfide p-nitrobenzoic acid（DTNB）Function generation 5-sulfhydryl-Nitrobenzoic acid（TNB）； TNBin 412nmThere is an absorption peak at the location, which was measured by 412 nmAbsorption rate increase, calculate AchEActivity.

Acetyldan alkaline esterase activity assay kit P450 series

composition

Reagent 3: Powder×1 Support,4℃preservation. Add before use 1.3 ml Reagent 2, thoroughly shake and dissolve.Reagent 4: Powder×1 Support,4℃preservation. Add before use 1.3 ml Reagent 2, thoroughly shake and dissolve.

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

Self provided instruments and supplies:

Visible spectrophotometer/ELISA reader, trace quartz colorimetric dish/96 Orifice plate, low-temperature centrifuge, water bath, adjustable pipette, and distilled water.

Crude enzymeLiquid extraction：

1. Organization: According to organizational quality（g）Reagent 1 volume(ml)for 1：5~10The proportion of（Suggest weighing the agreement 0.1gOrganize, join 1mlreagent1） Perform ice bath homogenization,8000g 4℃centrifugation 10minTake the supernatant for testing.

2. Bacteria and fungi: according to the number of cells（10⁴ One volume of reagent（ml）for 500~1000：1 The proportion of（suggestion 500 Ten Thousand Cells Plusenter 1ml Reagent 1），Ice bath ultrasonic disruption of cells（power 300w, Ultrasound 3 Seconds, intervals 7 Second, total time 3min）And then 8000g， 4℃, centrifugation 10minTake the supernatant and place it on ice for testing.

3. Serum and other liquids: direct measurement.

Measurement operation:

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. Take a trace quartz colorimetric dish/96 Orifice plates, sequentially added 20μlSupernatant160 μlReagent 210μl Reagent 3 and 10μlReagent 4, quickly mixUniform, in412nm Location determination3min Change in internal absorbance value, th10s The absorbance value is recorded asA1， 第190s The absorbance value is recorded asA2.△A Measurement tube=A2-A1. AchEActivity calculation:

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

1. organizationAchE activity

（1）Calculated based on protein concentration

Definition of active unit: every milligram of protein catalyzed per minute 1nmol TNB The enzyme quantity is 1 One enzyme activity unit.

AchE enzyme activity(nmol/min/mg prot)= (△A÷ε÷d×V Anti General×10⁹)÷（Cpr ×V sample）÷T

= 245×△A÷Cpr

（2）Calculated based on sample quality

Definition of active unit: catalytic production per gram of tissue per minute 1nmol TNB The enzyme quantity is 1 One enzyme activity unit.

AchE enzyme activity(nmol/min/g fresh weight)= (△A÷ε÷d×V Anti General×10⁹)÷（W ×V sample÷V Sample General）÷T

=245×△A÷W

2. Bacteria, cellsAchE activity

Definition of Active Unit: Each 10⁴ Cells catalyze production every minute 1nmol TNB The enzyme quantity is 1 One enzyme activity unit.

AchE enzyme activity(nmol/min/10⁴ cell)= (△A÷ε÷d×V Anti General×10⁹)÷(Number of cells)×V sample÷V Sample General）÷T

=245×△A÷cell count

3. serumAchE activity

Definition of active unit: catalytic production per milliliter of serum per minute 1nmol TNB The enzyme quantity is 1 One enzyme activity unit.

AchE enzyme activity(nmol/min /ml)= (△A÷ε÷d×V Anti General×10⁹)÷V sample÷T

=245×△A

ε：TNBMolar extinction coefficient,13.6×10³ L/mol/cm；dColor dish optical diameter,1 cm；VCounter total: total volume of the reaction system（L）， 200μl=2×10^-4L；V Sample total: volume of extraction solution,1 ml；10⁶：1mol=1×10⁶μmol；Cpr: Protein concentration（mg/ml）；V Sample: Add volume of supernatant（ml），0.02 ml；W : Sample quality;TResponse time（min），3 min.

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

1. organizationAchE activity

（1）Calculated based on protein concentration

Definition of active unit: every milligram of protein catalyzed per minute 1nmol TNB The enzyme quantity is 1 One enzyme activity unit.

AchE enzyme activity(nmol/min/mg prot)= (△A÷ε÷d×V Anti General×10⁹)÷（Cpr ×V sample）÷T

= 490×△A÷Cpr

（2）Calculated based on sample quality

Definition of active unit: catalytic production per gram of tissue per minute 1nmol TNB The enzyme quantity is 1 One enzyme activity unit.

AchE enzyme activity(nmol/min/g fresh weight)= (△A÷ε÷d×V Anti General×10⁹)÷（W ×V sample÷V Sample General）÷T

= 490×△A÷W

2. Bacteria, cellsAchE activity

Definition of Active Unit: Each 10⁴ Cells catalyze production every minute 1nmol TNB The enzyme quantity is 1 One enzyme activity unit.

AchE enzyme activity(nmol/min/10⁴ cell)= (△A÷ε÷d×V Anti General×10⁹)÷(Number of cells)×V sample÷V Sample General）÷T

=490×△A÷cell count

3. serumAchE activity

Definition of active unit: catalytic production per milliliter of serum per minute 1nmol TNB The enzyme quantity is 1 One enzyme activity unit.

AchE enzyme activity(nmol/min /ml)= (△A÷ε÷d×V Anti General×10⁹)÷V sample÷T

=490×△A

ε：TNB Molar extinction coefficient,13.6×10³ L/mol/cm；d：96 Aperture plate optical diameter,0.5 cm；V Counter total: total volume of the reaction system（L）， 200μl=2×10^-4L；VSample total: volume of extraction solution,1 ml；10⁶：1mol=1×10⁶μmol；Cpr: Protein concentration（mg/ml）；VSample: Add volume of supernatant（ml），0.02 ml；W : Sample quality;TResponse time（min），3 min.

Acetyldan alkaline esterase activity assay kit P450 series