Alkaline Phosphatase (AKP/ALP) Activity Assay Kit (DPP, Micro Method) - BioReagent, high purity

Cat. No.: A1492732

GRADE & PURITY BioReagent

Overview

Alkaline Phosphatase (AKP/ALP) is a zinc-containing glycoprotein enzyme capable of dephosphorylating corresponding substrates. It is widely distributed in various human organs. Under alkaline conditions, it can catalyze the hydrolysis of various substrates, including natural and synthetic phosphate monoester compounds, removing phosphate groups from substrate molecules via hydrolysis of phosphate monoesters to generate phosphate ions and free hydroxyl groups. Alkaline phosphatase can serve as a label enzyme for immunodiagnostic reagents and has important applications in the diagnosis and differentiation of skeletal and hepatobiliary system diseases.

Detection Principle: In an alkaline environment, AKP/ALP catalyzes the hydrolysis of disodium phenyl phosphate to generate free phenol. The phenol then reacts with 4-aminoantipyrine and potassium ferricyanide to form a red quinone derivative. This product has a characteristic absorption peak at 510 nm. The change in absorbance is used to characterize AKP/ALP activity.
Applicable Samples: Serum (plasma), animal tissues, urine.

A1492732	Component	48T	96T	Storage
A1492732A	Extraction Buffer	60 mL	120 mL	2-8℃
A1492732B	Chromogen A	2.5 mL	5 mL	2-8℃. Store in the dark.
A1492732C	Chromogen B	2.5 mL	5 mL	2-8℃. Store in the dark.
A1492732D	Chromogen C	7.5 mL	15 mL	2-8℃. Store in the dark.
A1492732E	Standard	0.5 mL	0.5 mL	2-8℃

Note: It is recommended to perform preliminary experiments using 2-3 samples expected to have significant differences before formal testing.

User-Provided Instruments and Reagents

Microplate reader or visible spectrophotometer (capable of measuring absorbance at 510 nm)

Incubator, Ice maker, Low-temperature centrifuge

96-well plate or micro glass cuvettes

Adjustable pipettes and tips

Deionized water

Homogenizer (for tissue samples)

Experimental Procedure

1. Reagent Preparation

Reagent Name	Reagent Preparation	Precautions
Extraction Buffer	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C.
Chromogen A	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C protected from light. Toxic; recommended to handle in a fume hood.
Chromogen B	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C protected from light.
Chromogen C	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C protected from light. Toxic; recommended to handle in a fume hood.
Standard	Ready-to-use; equilibrate to room temperature before use.	Store at 4°C. Irritating smell; recommended to handle in a fume hood.

2. Sample Preparation
Note: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 1 month.

2.1 Tissue Samples: Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Buffer, and homogenize on ice. Centrifuge at 10,000 rpm, 4°C for 10 minutes. Collect the supernatant and keep on ice for detection.
2.2 Serum (Plasma): Plasma and serum can be used directly for assay. EDTA and citrate should not be used as anticoagulants for plasma preparation; other anticoagulants are acceptable.
2.3 Urine: Detect directly.
For protein concentration determination, Aladdin BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) are recommended.

3. Assay Steps
3.1 Instrument Preparation: Preheat the microplate reader or visible spectrophotometer for at least 30 minutes. Set the wavelength to 510 nm. For spectrophotometers, zero the instrument with deionized water.
3.2 Assay Procedure: Add reagents and perform the reaction according to the table below:

Reagent	Blank Well (µL)	Standard Well (µL)	Control Well (µL)	Test Well (µL)
Deionized Water	4	0	0	0
Standard	0	4	0	0
Sample	0	0	0	4
Chromogen A	40	40	40	40
Chromogen B	40	40	40	40

Mix well and incubate at 37°C for 15 minutes.

Chromogen C	120	120	120	120
Sample	0	0	4	0

Mix well and measure the absorbance at 510 nm.
Note: The Standard and Blank wells need to be set up only once. Each sample requires a corresponding Control well.

Record the absorbances as A _blank , A _standard , A _control , and A _test .

4. Result Calculation
Note: We provide both derived and simplified calculation formulas, which are equivalent. The simplified formulas in bold are recommended as the final calculation formulas.

4.1 Based on Protein Concentration:

• Unit Definition: One unit of enzyme activity is defined as the amount that catalyzes the production of 1 μmol of phenol per minute per mg of protein at 37°C.**

• Formula:
  AKP/ALP (U/mg prot) = [C_standard × (A_test - A_control ) ÷ (A_standard - A_blank ) × V_sample ] ÷ (Cpr × V_sample ) ÷ T = 0.133 × (A_test - A_control ) ÷ (A _standard - A_blank ) ÷ Cpr

4.2 Based on Sample Fresh Weight:

• Unit Definition: One unit of enzyme activity is defined as the amount that catalyzes the production of 1 μmol of phenol per minute per gram of fresh tissue at 37°C.**

• Formula:
  AKP/ALP (U/g fresh weight) = [C_standard × (A_test - A_control ) ÷ (A_standard - A_blank ) × V_sample ] ÷ (W ÷ V_extract × V_sample ) ÷ T = 0.133 × (A_test - A_control ) ÷ (A_standard - A_blank ) ÷ W

4.3 Based on Liquid Volume:

• Unit Definition: One unit of enzyme activity is defined as the amount that catalyzes the production of 1 μmol of phenol per minute per mL of blood or urine at 37°C.**

• Formula:
  AKP/ALP (U/mL) = [C_standard × (A_test - A_control ) ÷ (A_standard - A_blank ) × V_sample ] ÷ V_sample ÷ T = 0.133 × (A_test - A_control ) ÷ (A_standard - A _blank )

Parameter Description:

• C_standard : Concentration of the standard, 2 μmol/mL

• V_sample : Volume of supernatant added to the reaction system, 0.004 mL

• T: Reaction time, 15 min

• V_extract : Volume of Extraction Buffer added, 1 mL

• W: Sample fresh weight, g

• Cpr: Protein concentration of the supernatant, mg/mL

Storage and Shipping

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Stability And Storage

Store at 2-8℃ long term (12 months). Store in the dark.