Alkaline phosphatase（AKP/ALP）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:

AKP/ALP It is a zinc containing sugar protease that can hydrolyze various natural and artificially synthesized phospholipid monoester compounds in alkaline environments.

AKP/ALP Widely distributed in various organs of the human body, mainly the liver.

Measurement principle:

In alkaline environments,AKP/ALP Catalyze the formation of free phenols from disodium phenylphosphate; Phenol and 4-The reaction between aminoantibilin and potassium ferrocyanide results in a red colorQuinone derivatives, in 510nmHas characteristic light absorption; by determination 510 nmCalculate the rate of increase in absorbanceAKPActivity.

Alkaline phosphatase activity assay kit esterase series

composition

Reagent 4: Liquid×1 Bottle,4℃Keep away from light and use until it turns blue-green. Standard: Liquid×1 Support（EP In the management,2 μmol/ml Phenol standard solution,4℃preservation.

Self provided instruments and supplies:

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

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

Extraction of crude enzyme solution:

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,4℃The8000gcentrifugation 10minTake the supernatant for testing.

2. Bacteria or cells: according to the number of bacteria or cells（104 One volume of reagent（ml）for 500~1000：1 The proportion of（suggestion 500 ten thousandCell addition 1mlReagent 1), ice bath ultrasonic disruption of cells（power 300w, Ultrasound 3 Seconds, intervals 7 Second, total time 3min）；then 8000g，4℃, centrifugation 10minTake the supernatant and place it on ice for testing.

3. Blood can be measured directly or after appropriate dilution.

Measurement steps:

1. spectrophotometer/Preheating of ELISA reader 30minAdjust the wavelength to 510 nmAdjust distilled water to zero.

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

3. Blank tube:取EP Tube, join in 4μl distilled water40μl Reagent 2,40μl Reagent 3, mix well and place in 37℃Insulation in water bath 15min；Add reagent four 120μlAfter mixing 510nmMeasure absorbance, denoted asABlank tube.

4. Standard tube:取EP Tube, join in 4μl Standard products,40μl Reagent 2,40μl Reagent 3, mix well and place in 37℃Insulation in water bath 15min；

Add reagent four 120μlAfter mixing 510nm Measure absorbance, denoted asA Standard tube.

5. Care for:取EP Tube, join in 4μl The supernatant,40μl distilled water40μl Reagent 3, mix well and place in 37℃Insulation in water bath 15min；Add reagent four 120μlAfter mixing 510nmMeasure absorbance, denoted asAMeasurement tube.

6. Measurement tube:取EP Tube, join in 4μl The supernatant,40μl Reagent 2,40μl Reagent 3, mix well and place in 37℃Insulation in water bath 15min；

Add reagent four 120μlAfter mixing 510nm Measure absorbance, denoted asA Measurement tube.Only one standard tube and blank tube need to be made, while each sample of the measuring tube and control tube needs to be made.

Note: Blank tubes and standard tubes only need to be measured once.

AKP/ALP Activity calculation:

1. In the bloodAKP/ALP Vitality Calculation

Definition of active unit:37℃Catalytic production per milliliter of blood per minute 1μmol Phenol is defined as 1 One enzyme activity unit.

AKP/ALP 活力(μmol/min /ml)= [C reference standard×(A Measurement tube-A To take care of)÷(A Standard tube-A Blank tube)×V Anti General] ÷V sample

÷T=6.8×(A Measurement tube-A To take care of)÷(A Standard tube-A Blank tube)

2. In organizations, bacteria, or cellsAKP/ALP Activity Calculation

（1）Calculated based on protein concentration

Definition of active unit:37℃Catalytic production per minute per milligram of protein 1 μ molPhenol is defined as 1 One enzyme activity unit. AKP/ALP(μmol/min/mg prot)=[C reference standard×(A Measurement tube-A To take care of)÷(A Standard tube-A Blank tube)×V Anti General]÷(Cpr×Vsample)÷T=6.8×(AMeasurement tube-ATo take care of)÷(AStandard tube-ABlank tube) ÷Cpr

（2）Calculated based on sample quality

Definition of active unit:37℃Catalytic production per minute per gram of tissue 1 M mol Phenol is defined as 1 One enzyme activity unit.

AKP/ALP(μmol/min/g fresh weight)=[C reference standard×(A Measurement tube-A To take care of)÷(A Standard tube-A Blank tube)×V Anti General]÷(W×V sample÷VSample General)÷T=6.8×(AMeasurement tube-ATo take care of)÷(AStandard tube-ABlank tube) ÷W

（3）Calculated based on the number of bacteria or cells

Definition of active unit:37℃Every middle 10⁴ Bacteria or cells catalyze production every minute 1 μ molPhenol is defined as 1 One enzyme activity unit.AKP/ALP (μmol/min/10)⁴ cell)=[C reference standard×(A Measurement tube-A To take care of)÷(A Standard tube-A Blank tube)×V Anti General]÷(cell count×V sample÷V Sample General)÷T= 6.8×(AMeasurement tube-ATo take care of)÷(AStandard tube-ABlank tube) ÷cell count

C Standard product:2μmol/ml；V Counter total: total volume of the reaction system（ml），205μl=0.205ml； CprCrude enzyme solution protein concentration（mg/ml），Additional testing is required, it is recommended to use the products produced by our company BCAProtein content determination kit;W : Sample quality;VSample: Volume of supernatant added to the reaction system（ml），0.004ml；VSample total: volume of extraction solution,1 ml；TResponse time（min），15 min.

Notes:

1. Reagents 2, 3, and 4 should be stored away from light.

2. After the reagent turns blue-green, it cannot be used again.

3. After adding reagent four, it must be mixed immediately, otherwise the color will not developcomplete.

Alkaline phosphatase activity assay kit esterase series