N-acetyl- β- D-glucosidase(NAG) Activity Assay Kit
enzyme labeling method
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The N-acetyl-β-D-glucosidase (NAG) Activity Assay Kit is widely used in biomedical research and clinical diagnostics to measure NAG enzyme activity, which serves as a biomarker for various conditions. It is particularly valuable in assessing kidney function, as elevated NAG levels in urine often indicate renal tubular damage or dysfunction. This kit is also employed in studies related to lysosomal storage disorders, where NAG activity is critical for understanding disease mechanisms. Additionally, it finds applications in environmental and food science to evaluate enzyme activity in samples, aiding in quality control and safety assessments. The kit provides a reliable, sensitive, and straightforward method for quantifying NAG activity, making it an essential tool in both research and diagnostic settings.
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N-acetyl-β-D-glucosidase (NAG) Activity Assay Kit - Microplate Method
Product Information
| Product Code | 111936 |
| Assay Format | Microplate method |
| Package Size | 100T |
Product Introduction
N-acetylglucosaminidase, also known as N-acetyl-β-D-glucosaminidase or NAG, is an intracellular lysosomal enzyme found in organs such as the kidney, liver, spleen, and brain. It is present at the highest level in renal proximal tubules.
NAG has a relatively large molecular weight and cannot be filtered by the glomerulus. When kidney damage occurs, NAG is released from cells into the renal tubules, causing urinary NAG to increase significantly. Urinary NAG activity is a highly sensitive and specific indicator of renal tubular lesions and can serve as an early indicator of renal tubular injury, with earlier predictive value than urinary albumin.
NAG hydrolyzes β-N-acetylglucosaminide to produce p-nitrophenol, which has a maximum absorption peak at 400 nm. NAG activity is calculated by measuring the rate of increase in absorbance.
Package Contents
| Code | Component | Quantity |
|---|---|---|
| 111936.1 | Reagent One | 1 bottle |
| 111936.2 | Reagent Two | 1 bottle |
| 111936.3 | Reagent Three | 1 bottle |
| 111936.4 | Extraction Solution | 1 bottle |
| 111936.5 | Standard | 1 vial |
| 111936.m | Manual | 1 copy |
Quality and Safety Information
| Raw Material or Packaging Name | Quality Standard | Main Toxicity |
|---|---|---|
| Reagent One | -- | -- |
| Reagent Two | -- | -- |
| Reagent Three | -- | -- |
| Extraction Solution | -- | -- |
| Standard | -- | -- |
Transportation and Storage
| Item | Condition |
|---|---|
| Transportation | Transport with ice packs. |
| Storage | Store Reagent One at -20°C. Store the remaining reagents at 2-8°C. |
| Shelf Life | 180 days |
Instructions for Use
1. Solution Preparation
- For Reagent One, add 2.5 mL distilled water before use and dissolve thoroughly. Store the remaining prepared reagent at -20°C.
- Before use, dilute the standard 8-fold to obtain a 0.625 umol/L standard solution.
2. Crude Enzyme Extract Preparation
Bacteria or Cultured Cells
- Collect the bacteria or cells into a centrifuge tube.
- Centrifuge and discard the supernatant.
- Add Extraction Solution according to the ratio of bacteria or cell number to Extraction Solution volume: 500-1000 × 104cells per 1 mL. It is recommended to add 1 mL Extraction Solution to 500 × 104bacteria or cells.
- Sonicate in an ice bath at 20% power or 200 W. Sonicate for 3 s, pause for 10 s, and repeat 30 times.
- Centrifuge at 15000 g and 4°C for 10 min.
- Collect the supernatant and place it on ice for testing.
Tissue
- Add Extraction Solution according to the ratio of tissue mass to Extraction Solution volume: 1 g tissue to 5-10 mL Extraction Solution. It is recommended to weigh approximately 0.1 g tissue and add 1 mL Extraction Solution.
- Homogenize in an ice bath.
- Centrifuge at 15000 g and 4°C for 10 min.
- Collect the supernatant and place it on ice for testing.
Serum or Plasma
Serum and plasma samples can be tested directly.
3. Assay Procedure
- Preheat the microplate reader for at least 30 min.
- Set the wavelength to 400 nm and zero with distilled water.
- Add the reagents sequentially into tubes or a 96-well plate according to the table below.
| Component | Assay Tube | Control Tube | Standard Tube | Blank Tube |
|---|---|---|---|---|
| Reagent I | 25 μL | |||
| Distilled Water | 25 μL | 25 μL | 35 μL | |
| Reagent II | 35 μL | 35 μL | 35 μL | 35 μL |
| Sample | 10 μL | 10 μL | ||
| Standard Solution | 10 μL |
Mix quickly and incubate at 37°C for 30 min.
| Component | Assay Tube | Control Tube | Standard Tube | Blank Tube |
|---|---|---|---|---|
| Reagent III | 130 μL | 130 μL | 130 μL | 130 μL |
Mix thoroughly and measure absorbance at 400 nm. Record the values as Aassay tube, Acontrol tube, Astandard tube, and Ablank tube.
Calculate ΔAassay= Aassay tube- Acontrol tube. Calculate ΔAstandard= Astandard tube- Ablank tube.
Each assay tube requires one control tube. The blank tube and standard tube only need to be run 1-2 times.
NAG Activity Calculation
1. Calculation by Protein Concentration
Unit definition: one enzyme activity unit is defined as the amount of enzyme that generates 1 nmol of p-nitrophenol per minute in the reaction system per mg of protein.
NAG (U/mg prot) = ΔAassay÷ (ΔAstandard÷ Cstandard) × 1000 × Vsample÷ (Cpr × Vsample) ÷ T = 20.83 × ΔAassay÷ ΔAstandard÷ Cpr
2. Calculation by Sample Mass
Unit definition: one enzyme activity unit is defined as the amount of enzyme that generates 1 nmol of p-nitrophenol per minute in the reaction system per g of sample.
NAG (U/g mass) = ΔAassay÷ (ΔAstandard÷ Cstandard) × 1000 × Vsample÷ (Vsample÷ Vtotal sample× W) ÷ T = 20.83 × ΔAmeasured÷ ΔAstandard÷ W
3. Calculation by Cell Number
Unit definition: one enzyme activity unit is defined as the amount of enzyme that produces 1 nmol of p-nitrophenol per minute in the reaction system per 104cells.
NAG (U/104cells) = ΔAmeasured÷ (ΔAstandard÷ Cstandard) × 1000 × Vsample÷ (cell number × Vsample÷ Vtotal sample) ÷ T = 20.83 × ΔAmeasured÷ ΔAstandard÷ cell number
4. Calculation by Liquid Volume
Unit definition: one enzyme activity unit is defined as the amount of enzyme that catalyzes the formation of 1 nmol of p-nitrophenol per minute in the reaction system per mL of liquid.
NAG (U/mL) = ΔAmeasured÷ (ΔAstandard÷ Cstandard) × 1000 × Vsample÷ Vsample÷ T = 20.83 × ΔAmeasured÷ ΔAstandard
Formula Parameters
| Symbol | Description |
|---|---|
| Cstandard | Concentration of the standard solution: 0.625 μmol/mL |
| Vtotal sample | Extraction Solution volume: 1 mL |
| Vsample | Sample volume added: 0.01 mL |
| Cpr | Protein concentration of the supernatant: mg/mL |
| T | Reaction time: 30 min |
| Cell number | Counted in ten-thousands |
| W | Sample mass: g |
| 1000 | Conversion factor: 1 μmol = 1000 nmol |
Precautions
- Before the formal assay, select 2-3 samples with large expected differences for a preliminary test.
- The following instruments and supplies must be provided by the user: microplate reader, benchtop centrifuge, water bath, adjustable pipettes, 96-well plate, mortar, ice, and distilled water.
- This 100T kit can test 48 samples.
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