Alkaline phosphatase (ALP) activity detection kit (pNPP, colorimetric method)

Cat. No.: A1509256

GRADE & PURITY BioReagent

Overview

Alkaline phosphatase (ALP or AKP) is a type of phosphatase widely distributed in mammalian tissues, with an optimal pH range of 9.2–9.8 for its activity. This enzyme is primarily located in areas of active substance exchange (cell membranes), such as the brush borders of intestinal epithelium and renal proximal tubules, the stereocilia of epididymal epithelium, the canalicular membranes of the liver, and the endothelium of arterioles and arterial capillaries. It is also found in the endoplasmic reticulum, Golgi apparatus, pinocytotic vesicles, lysosomes of intestinal epithelium, specific granules of neutrophils, and the cell membranes of smooth muscle.

Detection Principle

Para-nitrophenyl phosphate (pNPP) is a commonly used chromogenic substrate for phosphatases. Under alkaline conditions, it is hydrolyzed by alkaline phosphatase to produce p-nitrophenol. In an alkaline environment, p-nitrophenol converts to a quinoid structure, exhibiting a deep yellow color. The deeper the yellow color of the product, the higher the alkaline phosphatase activity, and vice versa. The absorbance is measured at 400–415 nm using spectrophotometry, allowing the calculation of alkaline phosphatase activity levels through colorimetric analysis.

Applicable Samples: Cell or tissue lysates or homogenates, plasma, serum, urine, etc.

A1509256	Component	60T	Storage
A1509256A	ALP Assay Buffer	50 mL	2-8℃
A1509256B	pNPP	2×1EA	-20℃. Store in the dark.
A1509256C	p-nitrophenol (10mM)	0.2 mL	-20℃. Store in the dark.
A1509256D	Stopping Solution	75 mL	RT.

Reagents, consumables and Equipments not provided

• Spectrophotometer (capable of measuring absorbance at 410 nm)
• Water bath or incubator
• Cuvettes

Operating Steps (for reference only)

1. Sample Preparation

1.1 Cell or Tissue Samples

Take an appropriate amount of cell or tissue lysate. If necessary, homogenize appropriately with PBS or physiological saline. Generally, use cell counts above 10⁶ or tissue amounts above 100 mg. Centrifuge at 3000–4000 g and collect the supernatant. Store at -20°C for alkaline phosphatase detection.

1.2 Plasma, Serum, and Urine Samples

Plasma and serum, prepared by conventional methods, can be directly used for detection with this kit. Urine can also typically be used directly. Store at -20°C. To eliminate interference from the sample's own color, set up a control containing plasma or serum but without the substrate.

1.3 High-Activity Samples

If the sample contains high alkaline phosphatase activity, dilute it with the original lysis buffer, PBS, or ALP Assay Buffer.

2. Preparation of Chromogenic Working Solution

Take one tube of pNPP, allow it to reach room temperature, and dissolve it in 15 mL of ALP Assay Buffer. Mix well and pre-cool on ice. The freshly prepared chromogenic working solution should be used within 6 hours.

3. Preparation of Standard Working Solution

Take the p-nitrophenol (10 mM), allow it to reach room temperature, and mix 0.1 mL with 1.9 mL of ALP Assay Buffer to obtain a concentration of 0.5 mM (p-nitrophenol, 0.5 mM). Store at -20°C. This solution is stable for 2 weeks. Further dilute the standard using p-nitrophenol (0.5 mM) according to the table below:

Tube Number	1	2	3	4	5	6
ALP Assay Buffer (mL)	0.45	0.4	0.3	0.25	0.1	0
p-nitrophenol (0.5 mM) (mL)	0.05	0.1	0.2	0.25	0.4	0.5
p-nitrophenol Concentration (μM)	50	100	200	250	400	500

4. ALP Sample Addition

Set up blank control, standard, and test tubes according to the table below. Add solutions in the specified order, taking care to avoid bubbles. If the alkaline phosphatase activity in the sample is too high, reduce the sample volume or dilute appropriately before measurement. It is recommended to set up duplicate tubes for sample detection.

Substance Added (mL)	Control Tube	Standard Tube	Test Tube
ALP Assay Buffer	0.5	—	—
Standard Working Solution (Tubes 1-6)	—	0.5	—
Sample	—	—	0.5
Chromogenic Working Solution	0.5	0.5	0.5

Mix well and incubate at 37°C for 20 minutes.

Stopping Solution

1

1

1

5. ALP Measurement

Zero the spectrophotometer with the control tube. Using a 1 cm light path cuvette, measure the absorbance at 410 nm. If 410 nm is not available, absorbance within the 400–415 nm range can be measured. Detection should generally be completed within 15 minutes.

6. Result Calculation

Definition of Alkaline Phosphatase Activity Unit: One unit of enzyme activity is defined as the amount of alkaline phosphatase required to hydrolyze the para-nitrophenyl phosphate chromogenic substrate to produce 1 micromole of p-nitrophenol per minute under pH 9.8 buffer conditions at 37°C.

When 0.5 mL of the standard from Tube 1 is added, its enzyme activity is 50 μM / 20 min = 2.5 U/L. Similarly, for p-nitrophenol concentrations of 100 μM, 200 μM, 250 μM, 400 μM, and 500 μM, the corresponding activities are 5, 10, 12.5, 20, and 25 U/L, respectively. Plot a standard curve with enzyme activity as the x-axis and the corresponding absorbance as the y-axis. Calculate the alkaline phosphatase activity in the sample based on the definition of enzyme activity.

Precautions

1. The test sample must not contain phosphatase inhibitors, and repeated freeze-thaw cycles should be avoided.

2. It is recommended to prepare a fresh standard curve for each assay to ensure accuracy. Also, avoid repeated freeze-thaw cycles of the standard.

3. If the measured sample value exceeds the upper limit of the standard curve, dilute the sample with ALP Assay Buffer and repeat the measurement.

4. One prepared batch of chromogenic working solution must be used on the same day. Therefore, prepare an appropriate number of samples for simultaneous detection.

5. p-nitrophenol solution is harmful to humans, and the stopping solution is corrosive. Handle with care.

6. For absolute quantification of enzyme activity, precise timing of the enzymatic reaction is necessary. In this case, incubating for 30 minutes or longer is recommended to minimize time errors during operation.

7. If the alkaline phosphatase activity in the sample is low, the incubation time can be appropriately extended to 30 minutes.

8. For your safety and health, please wear a lab coat and disposable gloves during operation.

9. Please use the reagent as soon as possible after opening to avoid affecting subsequent experimental results.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light,Room temperature,Store at -20°C

Shipped In

Ice chest + Ice pads

Stability And Storage

Each component has a shelf life of 1 year under corresponding storage conditions. A1509256A: Store at 2-8℃ long term (12 months). A1509256B, A1509256C: Store at -20℃ long term (12 months). Store in the dark. A1509256D: Store at room temperature long term

Contents & Storage

A1509256	Component	60T	Storage
A1509256A	ALP Assay Buffer	50 mL	2-8℃
A1509256B	pNPP	2×1EA	-20℃. Store in the dark.
A1509256C	p-nitrophenol (10mM)	0.2 mL	-20℃. Store in the dark.
A1509256D	Stopping Solution	75 mL	RT.