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

Cat. No.: A1518297

GRADE & PURITY BioReagent

Descripción general

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

• Microplate reader (capable of measuring absorbance at 405 nm)

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

• Centrifuge, water bath or incubator

• PBS or normal saline

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. Carefully open the vial, add 0.25 mL of deionized water, close the cap tightly, and dissolve completely by vortexing. Keep on ice until use. Any unused portion can be stored at -20°C protected from light for up to 2 days. To prepare the Working Color Solution, dilute the dissolved pNPP with TRAP Assay Buffer at a ratio of 1:20. Mix well, keep on ice, and protect from light. The freshly prepared Working Color Solution is stable for 6 hours.

3. Preparation of Standard Curve Dilutions

Allow the p-nitrophenol (10 mM) to equilibrate to room temperature. Dilute it with TRAP Assay Buffer at a ratio of 1:19 to achieve a concentration of 0.5 mM. Then, prepare serial dilutions according to the table below:

Addition (μL)	1	2	3	4	5	6
p-nitrophenol (0.5 mM)	2	4	8	12	16	20
TRAP Assay Buffer	18	16	12	8	4	0
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 wells in a 96-well plate as described below. Add reagents 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.

Addition (μL)	Blank Well	Standard Wells	Test Well	Control Well
TRAP Assay Buffer	20	—	—	40
Serial Standards (1~6)	—	20	—	—
Test Sample	—	—	20	20
Working Color Solution	40	40	40	—
Tartrate Solution	4	4	4	4

Note: The Control well corrects for background absorbance from the sample itself and non-specific reactions. For final calculations, subtract the Control well absorbance from the Test well absorbance (ΔA₄₀₅).

5. Measurement

Mix gently. Incubate at 37°C for 10 minutes. Stop the reaction by adding 160 μL of Stopping Solution to each well. Measure the absorbance of each well at 405 nm using a microplate reader.

6. Calculation of Results

Plot the standard curve using the concentrations of the serial standards (0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mM) as the x-axis and their corresponding absorbance values (Standard well OD - Blank well OD) as the y-axis. Perform linear regression to obtain the equation y = ax + b.

Determine the amount of p-nitrophenol generated in the Test sample, x (in mM), from the standard curve using the formula x = (ΔA₄₀₅- b) / a.

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. Test samples must not contain phosphatase inhibitors. Avoid repeated freeze-thaw cycles.

2. If the sample volume is limited, reduce the sample addition volume and adjust the total volume to 20 μL with TRAP Assay Buffer.

3. If measurement at 405 nm is not possible, absorbance in the range of 400-415 nm is also acceptable.

4. It is recommended to generate a standard curve for each assay for optimal accuracy. Avoid repeated freeze-thaw cycles of the standard.

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

6. When assaying tissue or cell samples, total protein concentration must be determined. A BCA protein assay kit is recommended.

7. Prepare only enough Working Color Solution for same-day use. It is advisable to plan experiments to assay multiple samples together.

8. p-nitrophenol solution is harmful. Stopping Solution is corrosive. Please handle with care. Wear laboratory coat and disposable gloves for safety and health protection.

9. For absolute quantification of enzyme activity, precise timing of the enzymatic reaction is critical. Incubating for 30 minutes or longer is recommended to minimize timing errors.

10. This product is for research use only and is not intended for diagnostic or therapeutic applications.

Almacenamiento y envío

Condiciones de almacenamiento de almacenamiento

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

Enviado en

Ice chest + Ice pads

Estabilidad y almacenamiento

Each component has a shelf life of 1 year under corresponding storage conditions. A1518297A, A1518297C: Store at 2-8℃ long term (12 months). A1518297B, A1518297D: Store at -20℃ long term (12 months). Store in the dark. A1518297E: Store at room temperature

Contents & Storage

A1518297	Component	120T	Storage
A1518297A	TRAP Assay Buffer	30 mL	2-8℃.
A1518297B	pNPP	1 EA ×2	-20℃. Store in the dark.
A1518297C	Tartrate Solution	1 mL	2-8℃.
A1518297D	p-nitrophenol (10mM)	0.5 mL	-20℃. Store in the dark.
A1518297E	Stopping Solution	20 mL	RT.