Acid Phosphatase (ACP) Activity Assay Kit (pNPP, Micro Method)

Cat. No.: A1509019

GRADE & PURITY BioReagent

Overview

Acid phosphatase (ACP) is widely distributed across various tissues. It is primarily located within the lysosomes of cells, often serving as a lysosomal marker enzyme. Extralysosomal ACP is found in the endoplasmic reticulum and cytoplasm. ACP varies among different animal species, with an optimal pH range of 4.5–5.5. ACP is a protein family; in mammals, its molecular weight ranges from 18 kD to 100 kD. This enzyme is classified into two types: tartrate-sensitive and fluoride-sensitive. Lysosomal ACP is tartrate-sensitive, while ACP in red blood cells and macrophages is fluoride-sensitive. The normal activity range of ACP in plasma is 2–7.9 U/L, and in serum it is 2.5–11.7 U/L. Semen contains a high concentration of acid phosphatase, with activity reaching 87–436 KU/L.

Detection Principle:

Para-nitrophenyl phosphate (pNPP) is a commonly used chromogenic substrate for phosphatases. Under acidic conditions, pNPP is hydrolyzed by acid phosphatase to generate p-nitrophenol. p-Nitrophenol turns yellow under alkaline conditions. The deeper the yellow color, the higher the ACP activity, and vice versa. By measuring the absorbance at 400–415 nm, the ACP activity level can be calculated via colorimetric analysis.

Applicable Samples: Lysates or homogenates of cells or tissues, plasma, serum, urine, etc.

Reagents, consumables and Equipments not provided

• Microplate reader (capable of measuring absorbance at 410 nm)

• 96-well plate, adjustable pipettes and tips, centrifuge tubes

• Centrifuge, water bath or incubator

Procedure (For Reference Only)

1. Sample Preparation

1.1 Plasma, Serum, and Urine Samples: Plasma and serum prepared by standard procedures can be directly used for assay. Urine can also typically be used directly. Store at -20°C for ACP detection.

1.2 Cell or Tissue Samples: Take an appropriate amount of cell or tissue lysate. Homogenize with PBS or saline if necessary (generally >10⁶ cells or >100 mg tissue). Centrifuge at 3000–4000 g, collect supernatant. Store at -20°C for ACP detection.

1.3 Plant Samples: Take appropriate plant tissue, add a small amount of saline or PBS, thoroughly crush or grind. Let stand for 30 min, filter through gauze or filter paper. Centrifuge at 4000 g for 20 min, collect supernatant and measure volume. Store at -20°C for ACP detection.

1.4 High-Activity Samples: If the sample contains high ACP activity, dilute it with the original lysis buffer, PBS, or ACP Assay Buffer.

2. Chromogenic Working Solution Preparation

Take one vial of pNPP, bring to room temperature, and dissolve it in 2.5 mL of ACP Assay Buffer. Mix well and pre-cool on ice. The newly prepared working solution should be used within 6 hours.

3. Standard Working Solution Preparation

Take the p-nitrophenol (10 mM), bring to room temperature. Pipette 10 µL and dissolve it in 190 µL of ACP Assay Buffer to achieve a concentration of 0.5 mM (p-nitrophenol (0.5 mM)). This reagent is stable at -20°C for 2 weeks. Further dilute the standard using p-nitrophenol (0.5 mM) as per the table below:

Added (µL)	Std.1	Std.2	Std.3	Std.4	Std.5	Std.6
ACP Assay Buffer	90	80	60	50	20	0
p-nitrophenol (0.5mM)	10	20	40	50	80	100
p-nitrophenol Conc. (µM)	50	100	200	250	400	500

4. ACP Assay Setup

Set up the control, standard, and assay wells according to the table below. Add solutions in the specified order, avoiding bubble formation. If sample ACP activity is too high, reduce sample volume or dilute appropriately before assay. Assay samples in duplicate if possible.

Added (µL)	Control Well	Standard Well	Assay Well
ACP Assay Buffer	40	—	—
Standard Working Solution (Std.1-6)	—	40	—
Sample to be Tested	—	—	40
Chromogenic Working Solution	40	40	40

Mix well, incubate at 37°C for 25–30 min.

Stopping Solution

160

160

160

5. ACP Measurement

Using the control well to zero the instrument, measure the absorbance of standard and assay wells at 410 nm (recorded as A_Standard and A_Assay). If 410 nm is unavailable, absorbance between 400–415 nm is acceptable. Complete measurements within 15 min.

6. Calculation of Results

Definition of ACP Activity Unit: One unit of enzyme activity is defined as the amount of acid phosphatase required to hydrolyze the para-nitrophenyl phosphate chromogenic substrate to produce 1 micromole of p-nitrophenol per minute under the conditions of pH 4.8 buffer at 37°C.

When adding the substance from Standard Tube 1 (0.3 mL equivalent), the enzyme activity is 50 µM/20 min = 2.5 U/L. By analogy, adding p-nitrophenol at concentrations of 100 µM, 200 µM, 250 µM, 400 µM, and 500 µM corresponds to activities of 5, 10, 12.5, 20, and 25 U/L, respectively.

Plot a standard curve with enzyme activity (U/L) on the X-axis and corresponding absorbance on the Y-axis. Calculate the acid phosphatase activity in the sample based on the enzyme activity definition.

Precautions

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

2. It is recommended to perform a standard curve with each assay for accuracy. Avoid repeated freeze-thaw cycles of the standard.

3. If a microplate reader is unavailable, a standard spectrophotometer can be used. Consider the minimum detection volume of the cuvette and use a small-volume cuvette if possible.

4. If the sample value exceeds the upper limit of the standard curve, dilute the sample with ACP Assay Buffer and repeat the assay.

5. One vial of prepared chromogenic working solution must be used on the same day. Plan to test an appropriate number of samples together.

6. p-Nitrophenol solution is harmful to humans. The stopping solution is corrosive. Handle with care.

7. For absolute quantification of enzyme activity, time the reaction precisely. Incubating for 30 min or longer is recommended to minimize timing errors during operation.

8. If sample ACP activity is low, the incubation time can be appropriately extended to 60 min.

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. A1509019A: Store at 2-8℃ long term (12 months). A1509019B, A1509019C: Store at -20℃ long term (12 months). Store in the dark. A1509019D: Store at room temperature long term

Contents & Storage

A1509019	Component	120T	Storage
A1509019A	ACP Assay buffer	15 mL	2-8℃.
A1509019B	pNPP	2×1EA	-20℃. Store in the dark.
A1509019C	p-nitrophenol (10mM)	0.3 mL	-20℃. Store in the dark.
A1509019D	Stopping Solution	20 mL	RT.