Carboxylesterase（CarE）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:

mammalCarE, also known as lipophilic esterase（aliesterase）Widely distributed in tissues and organs, it belongs to the serine acid hydrolase family.CarECatalyze the hydrolysis of endogenous and exogenous substances containing ester bonds, amide bonds, and thioester bonds, but cannot catalyze the hydrolysis of acetyl bases and their analogues.CarE Involved in lipid transport and metabolism, and related to detoxification and metabolism of various drugs, environmental toxins, and carcinogens, organophosphate pesticides can be combined andinhibitionCarEActivity.

Measurement principle:

CarE Can catalyze acetic acid-1-Naphthalene ester generates naphthalene ester, and solid basket color development occurs; in 450 nm Rate of increase in light absorption, calculationCarE Activity.

Carboxyesterase (CarE) activity assay kit

composition

Reagent 3: Powder×2 Support, 4℃Save, retrieve before use 1 Reagent three, add 0.6ml Dissolve completely in anhydrous ethanol; Reagent 4: Powder×2Support,-20℃Save, retrieve before use 1 Add a small amount of reagent 2 to dissolve reagent 4;

Preparation of working fluid: prepare before use, towards 1 Add dissolved reagent three and reagent four to bottle reagent two 1 Support, fully dissolve, filter,4℃Store away from light, usable 1 week

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

Self provided instruments and supplies:

Visible spectrophotometer, desktop centrifuge, adjustable pipette1 ml Glass colorimetric dish, mortar, ice, and distilled water.

Extraction of crude enzyme solution:

Preparation of bacterial and cellular samples

Collect bacteria or cells into centrifuge tubes, centrifuge and discard the supernatant; According to each 200 Ten thousand bacteria or cells added 400μl Reagent 1: Ultrasonic disruption of bacteria or cells（power 20%, Ultrasound 3s, interval 10sRepeat, repeat 30 Secondary);12000g 4℃centrifugation 30minTake the supernatant for testing.

2Organization: According to the quality of the organization（g）Reagent 1 volume(ml)for 1：5~10 The proportion of（Suggest weighing the agreement 0.1g Organize, join 1ml Reagent 1) Perform ice bath homogenization;12000g 4℃centrifugation 30minTake the supernatant for testing.

3Liquid: direct measurement

Measurement operation:

1. spectrophotometer/Preheating of ELISA reader 30minAdjust the wavelength to 450 nmAdjust distilled water to zero.

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

3. Blank tube:Take a trace glass colorimetric dish/96 Orifice plates, sequentially added 5μl Distilled water and 200μl Reagent 2, mix quickly and thoroughly 450nm Test locationfixed 3minChange in internal absorbance value, th 10sThe absorbance value is recorded asA1， 第 190sThe absorbance value is recorded asA2.△ABlank tube=A2-A1

4. Measurement tube:Take a trace glass colorimetric dish/96 Orifice plates, sequentially added 5μl The supernatant and 200μl Reagent 2, mix quickly and thoroughly 450nm Test location

fixed 3min Change in internal absorbance value, th 10s The absorbance value is recorded asA3， 第 190s The absorbance value is recorded asA4.△A Measurement tube=A4-A3

Note: Blank tubes only need to be measured once.

CarE Activity calculation formula:

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

1Within the organizationCarE activity

（1）Calculated by protein concentration

CarE Definition of Active Unit: Eachmg Tissue proteins are present 37℃The catalytic absorbance value increases every minute in the reaction system 1 defined as 1 One enzyme activity unit.

CarE enzyme activity(U/mg prot)= (△A Measurement tube-△A Blank tube)×V Anti General÷(Cpr×V sample)÷T

=13.67×(△A Measurement tube-△A Blank tube)÷Cpr

（2）Calculated based on the fresh weight of the sample

CarE Definition of Active Unit: Eachg The organization is 37℃The catalytic absorbance value increases every minute in the reaction system 1 defined as 1 One enzyme activity unit.

CarE enzyme activity(U/g fresh weight)= (△A Measurement tube-△A Blank tube)×V Anti General×（V Sample General÷V sample）÷W÷T

=13.67×(△A Measurement tube-△A Blank tube)÷W 2In bacteria or cellsCarEactivity

CarE Definition of Active Unit: Each 1 Ten thousand bacteria or cells are present 37℃The catalytic absorbance value increases every minute in the reaction system 1 Defined as aCarE live

Sexual units.

CarE enzyme activity(U/10⁴ cell)= (△A Measurement tube-△A Blank tube) ×V Anti General×（V Sample General÷V Sample)÷Cell density（10⁴ cell/ml）

÷T=13.67×(△A Measurement tube-△A Blank tube)÷Cell density（10⁴ cell/ml） 3In liquidCarEactivity

CarE Definition of active unit: per milliliter of sample 37℃The catalytic absorbance value increases every minute in the reaction system 1 defined as 1 One enzyme activity unit.

CarE enzyme activity(U/ml )= (△A Measurement tube-△A Blank tube)×V Anti General÷V sample÷T

=13.67×(△A Measurement tube-△A Blank tube)

V Counter total: the total volume of the reaction system,205μl=2.05×10^-4L；V Sample total: total volume of supernatant,1 ml；V Sample: Add volume of supernatant(ml)，

0.005 ml；CprProtein concentration,mg/ml；W: Sample quality（g）；TResponse time（min），3min.

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

1Within the organizationCarE activity

（3）Calculated by protein concentration

CarE Definition of Active Unit: Eachmg Tissue proteins are present 37℃The catalytic absorbance value increases every minute in the reaction system 1 defined as 1 One enzyme activity unit.

CarE enzyme activity(U/mg prot)= (△A Measurement tube-△A Blank tube)×V Anti General÷(Cpr×V sample)÷T

=13.67×(△A Measurement tube-△A Blank tube)÷Cpr

（4）Calculated based on the fresh weight of the sample

CarE Definition of Active Unit: Eachg The organization is 37℃The catalytic absorbance value increases every minute in the reaction system 1 defined as 1 One enzyme activity unit.

CarE enzyme activity(U/g fresh weight)= (△A Measurement tube-△A Blank tube) ×V Anti General×（V Sample General÷V sample）÷W÷T

=13.67×(△A Measurement tube-△A Blank tube)÷W 2In bacteria or cellsCarEactivity

CarE Definition of Active Unit: Each 1 Ten thousand bacteria or cells are present 37℃The catalytic absorbance value increases every minute in the reaction system 1 Defined as aCarE live

Sexual units.

CarE enzyme activity(U/10⁴ cell)= (△A Measurement tube-△A Blank tube)×V Anti General×（V Sample General÷V Sample)÷Cell density（10⁴ cell/ml）

÷T=13.67×(△A Measurement tube-△A Blank tube)÷Cell density（10⁴ cell/ml）

3. In liquidCarE activity

CarE Definition of active unit: per milliliter of sample 37℃The catalytic absorbance value increases every minute in the reaction system 1 defined as 1 One enzyme activity unit.

CarE enzyme activity(U/ml )= (△A Measurement tube-△A Blank tube)×V Anti General÷V sample÷T

=13.67×(△A Measurement tube-△A Blank tube)

V Counter total: the total volume of the reaction system,205μl=2.05×10^-4L；V Sample total: total volume of supernatant,1 ml；V Sample: Add volume of supernatant(ml)，

0.005 ml；CprProtein concentration,mg/ml；W: Sample quality（g）；TResponse time（min），3min.

Carboxyesterase (CarE) activity assay kit