Tartrate Resistant Acid Phosphatase (TRAP) Activity Assay Kit (pNPP, Colorimetric Method)

Cat. No.: A1518291

GRADE & PURITY BioReagent

Overview

Acid phosphatase (ACP) is a widely distributed enzyme found in various tissues throughout the body. It is primarily localized in cellular lysosomes and is therefore commonly used as a lysosomal marker enzyme. Additionally, ACP is present in the endoplasmic reticulum and cytoplasm. The optimal pH range for this enzyme is 4.5 to 5.5, and its characteristics vary among different animal species.

Tartrate-resistant acid phosphatase (TRAP) is a specific isoform of ACP. It is found within cytoplasmic vacuoles in normal human alveolar macrophages and in the spleen of patients with leukemia, and is not typically released into the bloodstream. TRAP in the blood originates mainly from osteoclasts. Therefore, measuring blood TRAP levels can reflect osteoclast activity and is widely regarded as the only hematological indicator for assessing bone resorption activity in the body. TRAP is a glycosylated, metal-containing enzyme highly expressed in osteoclasts and chondroclasts, and is also expressed in activated macrophages and neurons.

Assay Principle

Under acidic conditions, ACP acts on p-nitrophenyl phosphate (pNPP), hydrolyzing it to form p-nitrophenol (p-NP). In an alkaline environment, p-NP produces a yellow color, with the intensity being directly proportional to the enzyme activity, showing maximum absorbance at 405 nm. By adding an appropriate amount of tartrate to the reaction system, the activity of other ACP isoforms is inhibited, and the measured ACP activity specifically represents TRAP activity. TRAP activity levels are calculated by measuring the amount of p-NP generated.

Applicable Samples

Cell or tissue lysates, homogenates, plasma, serum, urine, etc.

Reagents, consumables and Equipments not provided

• Spectrophotometer capable of measuring absorbance at 405 nm
• 1.0 mL cuvettes, centrifuge tubes
• Centrifuge, water bath or incubator
• PBS or physiological saline, deionized water

Assay Procedure (For Reference Only)

1. Sample Preparation

1.1 Plasma, Serum, and Urine Samples

Plasma and serum prepared by routine methods can be used directly for the assay. Urine can also typically be used directly. If not assayed immediately, samples can be stored at -80°C but avoid repeated freeze-thaw cycles.

1.2 Tissue Samples

Homogenize tissue samples using a ratio of tissue weight (g) : normal saline volume (mL) = 1:10. Centrifuge and collect the supernatant for analysis. Retain an aliquot of the supernatant for BCA protein concentration determination.

1.3 Cell Samples

Homogenize cell samples using a ratio of cell number (10⁶) : normal saline volume (mL) = 5:1. Centrifuge and collect the supernatant for analysis. Retain an aliquot of the supernatant for BCA protein concentration determination.

Note: Before formal testing, perform a preliminary experiment using 2-3 samples with expected activity differences, diluted to various concentrations. Based on the preliminary results and the kit's linear range (5.0-300 U/L), dilute samples appropriately with PBS or normal saline before the final assay.

2. Preparation of Working Color Solution

Briefly centrifuge one vial of pNPP before opening. Carefully open the vial, add 0.5 mL of deionized water, cap tightly, and vortex until completely dissolved. Keep on ice until use. Any unused portion can be stored at -20°C protected from light for up to 2 days.

Mix the dissolved pNPP with TRAP Assay Buffer at a ratio of 1:20 (e.g., for 1 part pNPP solution, add 19 parts TRAP Assay Buffer). Vortex to mix. This freshly prepared Working Color Solution is stable for 6 hours when kept on ice and protected from light.

3. Preparation of Standard Curve Dilutions

Allow the p-nitrophenol (10mM) to reach room temperature. 

Prepare a 0.5 mM intermediate standard by mixing p-nitrophenol (10mM) with TRAP Assay Buffer at a ratio of 1:19 (e.g., 0.01 mL p-nitrophenol + 0.19 mL TRAP Assay Buffer).

Prepare serial dilution standards in tubes or a microplate according to the table below:

Additive (mL)	1	2	3	4	5	6
p-nitrophenol (0.5 mM)	0.01	0.02	0.04	0.06	0.08	0.10
TRAP Assay Buffer	0.09	0.08	0.06	0.04	0.02	0.00
Standard Concentration (mM)	0.05	0.1	0.2	0.3	0.4	0.5

4. TRAP Assay Setup

Set up Blank, Standard, Test, and Control tubes as per the table below. Add solutions in the order listed, avoiding bubbles.

If sample TRAP activity is expected to be very high, reduce the sample volume or dilute the sample appropriately. It is recommended to run samples in duplicate or triplicate.

Additive (mL)	Blank Tube	Standard Tube (1-6)	Test Tube	Control Tube
TRAP Assay Buffer	0.1	—	—	0.2
Series Standards (1-6)	—	0.1	—	—
Sample	—	—	0.1	0.1
Working Color Solution	0.2	0.2	0.2	—
Tartrate Solution	0.02	0.02	0.02	0.02

Note: The Control tube is used to correct for the background absorbance contributed by the sample itself and non-specific reactions. For the final calculation, use the absorbance difference: ΔA₄₀₅ = (Test Tube A₄₀₅) - (Control Tube A₄₀₅).

5. Measurement

Mix gently. Incubate at 37°C for exactly 10 minutes.

Add 0.8 mL of Stopping Solution to each tube to terminate the reaction.

Zero the spectrophotometer with the Blank tube at 405 nm using a 1 cm path length cuvette.

Measure the absorbance (A₄₀₅) of all tubes (Standard, Test, Control).

6. Calculation of Results

Standard Curve: Plot the absorbance (A₄₀₅) of the Standard tubes (Y-axis) against their corresponding concentrations (0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mM) (X-axis). Perform linear regression to obtain the standard curve equation: y = ax + b.

Calculate p-NP Concentration (x) for Samples: Using the ΔA₄₀₅ value (Test A₄₀₅ - Control A₄₀₅) for the sample, calculate the concentration of p-nitrophenol generated: x = (ΔA₄₀₅- b) / a (unit: mM).

Definition of Enzyme Activity Unit

• For Tissue and Cell Samples: One unit of TRAP activity is defined as the amount of enzyme that catalyzes the production of 1 μmol of p-nitrophenol per minute per gram of protein at pH 4.8 and 37°C.

  • Formula: TRAP Activity (U/g prot) = x×1000×N÷t÷Cpr

• For Serum (Plasma) and Urine Samples: One unit of TRAP activity is defined as the amount of enzyme that catalyzes the production of 1 μmol of p-nitrophenol per minute per liter of serum (plasma) or urine at pH 4.8 and 37°C.

  • Formula: TRAP Activity (U/L) = x×1000×N÷t

Parameter Description:

• a: Slope of the standard curve

• b: Intercept of the standard curve

• N: Dilution factor of the sample before addition to the reaction

• 1000: Unit conversion factor (1 mmol/L = 1000 μmol/L)

• Cpr: Protein concentration of the sample (g prot/L)

• t: Enzymatic reaction time (min)

• ΔA₄₀₅: Absolute OD of the sample (Test OD - Control OD)

• y: Standard OD - Blank OD

Notes

1. Sample Integrity: Test samples must not contain phosphatase inhibitors. Avoid repeated freeze-thaw cycles.

2. Low Sample Volume: If the sample volume is limited, reduce the sample volume and adjust the total volume to 0.1 mL with TRAP Assay Buffer in the Test and Control tubes.

3. Wavelength: If measuring at 405 nm is not possible, absorbance in the range of 400-415 nm is acceptable.

4. Standard Curve: It is recommended to generate a standard curve for each assay for the most accurate results. Avoid repeated freeze-thaw cycles of the p-nitrophenol standard.

5. Low Activity Samples: If sample TRAP activity is expected to be low, the incubation time can be extended up to 30 minutes.

6. Protein Assay: For tissue and cell samples, total protein concentration must be determined. A BCA Protein Assay Kit is recommended.

7. Working Solution Stability: Prepare only the amount of Working Color Solution needed for the immediate experiments, as it should be used on the day of preparation.

8. Safety: The p-nitrophenol solution is harmful to humans. The Stopping Solution is corrosive. Please handle with care.

9. Timing for Absolute Quantification: For precise absolute quantification, ensure accurate timing of the enzyme reaction. Using a longer incubation time (e.g., 30 min) can help minimize timing errors.

10. Personal Protection: For your safety and health, please wear laboratory appropriate clothing and disposable gloves.

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

各组分在相应的储存条件下保质期1年。

Contents & Storage

A1518291	Component	100T	Storage
A1518291A	TRAP Assay Buffer	50 mL	2-8℃.
A1518291B	pNPP	1 EA ×2	-20℃. Store in the dark.
A1518291C	Tartrate Solution	2.5 mL	2-8℃.
A1518291D	p-nitrophenol (10mM)	0.5 mL	-20℃. Store in the dark.
A1518291E	Stopping Solution	85 mL	RT.