Solarbio Tissue and Blood Alkaline Phosphatase (AKP/ALP) Activity Assay Kit

Tissue and Blood Alkaline Phosphatase (AKP/ALP) Activity Assay Kit

Note: Take two or three different samples for prediction before test.

Operation Equipment: Spectrophotometer

Catalog Number: BC2140

Size: 50T/24S

Components:

Extraction reagent: Liquid 30 mL×1 bottle, storage at 4℃.

Reagent 1: Liquid 10 mL×1 bottle, storage at 4℃ and protect from light.

Reagent 2: Liquid 10 mL×1 bottle, storage at 4℃ and protect from light.

Reagent 3: Liquid 30 mL×1 bottle, storage at 4℃ and protect from light. It cannot be used if it turns to blue-green.

Standard: Liquid 1 mL×1 bottle, 10 μmol/mL phenol standard solution, storage at 4℃. Dilute with distilled water to 2.5 μmol/mL before use.

Product Description:

AKP/ALP is a zinc-containing glycoprotease, which hydrolysis various natural and synthetic phospholipid monoester compounds in alkaline condition. AKP / ALP are widely distributed in human organs, mainly in liver.

In alkaline condition, AKP/ALP catalyzes hydrolysis of disodium phenyl phosphate to phenol, and the phenol reacts with 4-Aminoantipyrine and potassium ferricyanide to form red quinone derivative, which can be detected absorbance at 510 nm. AKP/ALP activity can be calculated by measuring the absorbance increase rate at 510 nm.

Reagents and Equipment Required but Not Provided:

Spectrophotometer, desk centrifuge, transferpettor, 1 mL glass cuvette, ice and distilled water.

Procedure:

I. Enzyme preparation:

Add 1 mL Extraction reagent to 0.1 g tissue, grind thoroughly. Centrifuge at 4℃, 10000 rpm for 10 minutes. Take the supernatant on the ice for test. Blood sample can be detected directly. Dilute with Extraction reagent if concentrate is high.

II. Determination procedure:

1. Preheat spectrophotometer for 30 minutes, adjust the wavelength to 510 nm, set the counter to zero with distilled
2. Add reagents in 1.5 mL cuvette as the following:

III. AKP/ALP activity calculation:

• Protein concentration:

Unit definition: One unit of enzyme activity is defined as the amount of enzymes catalyzes the generation of 1 μmol phenol in the reaction system per minute at 37℃ every mg protein.

AKP/ALP (U/mg prot) =[C×(A3-A4)÷(A1-A2)×Vs]÷(Cpr×Vs)÷T=0.167×(A3-A4)÷(A1-A2)÷Cpr

• Sample weight:

Unit definition: One unit of enzyme activity is defined as the amount of enzymes catalyzes the generation of 1 μmol phenol in the reaction system per minute at 37℃ every g sample.

AKP/ALP (U/g weight) =[C×(A3-A4)÷(A1-A2)×Vs]÷(W÷Ve×Vs)÷T=0.167×(A3-A4)÷(A1-A2)÷W

• Blood:

Unit definition: One unit of enzyme activity is defined as the amount of enzymes catalyzes the generation of 1 μmol phenol in the reaction system per minute at 37℃ every mL serum.

AKP/ALP (U/mL) =[C×(A3-A4)÷(A1-A2)×Vs]÷Vs÷T=0.167×(A3-A4)÷(A1-A2)

C: Standard concentration, 2.5 μmol/mL;

Vrv: Total reaction volume, 1020 μL=1.02 mL; Vs: Supernatant volume, 0.02 mL;

Ve: Extraction volume, 1 mL; W: Sample weight, g;

T: Reaction time, 15 minutes;

Cpr: Sample protein concentration, mg/mL.

Note:

1. Reagent 1, Reagent 2 and Reagent 3 should be protected from
2. Reagent 3 cannot be used if it has changed toblue-green.
3. Mix thoroughly quickly after adding Reagent 3 to avoid incomplete

Experimental example:

1. Take 0.1g of mouse pancreas and add 1 mL of Extract solution for homogenate. After taking the supernatant, operate according to the determination steps. Calculate AT = 0.324, AC = 0.037, AB = 0.022,  AS = 0.677. Calculate the enzyme activity according to the sample mass: AKP/ALP enzyme activity (U/g mass) = 0.167×(AT-AC)÷(AS-AB) ÷ W =0.167×(0.324-0.037)÷(0.677-0.022)÷0.1=0.732 U/g
2. After taking the rabbit serum, the operation is carried out according to the determination steps, and the enzyme activity is calculated as follows: AT = 0.190, AC = 0.015, AB = 0.022, AS = 0.677. According to the blood volume, the enzyme activity is calculated as follows: AKP/ALP enzyme activity (U/mL) = 0.167×(AT-AC) ÷ (AS-AB) =0.167×(0.190-0.015)÷(0.677-0.022)=0.0446U/mL.

Related Products：

BC2020/BC2025 Acetylcholinesterase(AchE) Activity Assay Kit

BC2130/BC2135 Acid Phosphatase(ACP) Activity Assay Kit

BC0840/BC0845 Carboxylesterase(CarE) Activity Assay Kit