N-acetyl-β-D-glucosidase (NAG) Activity Assay Kit - Micro Method

Product code: 111935

Product Introduction

NAG is an acidic hydrolase found in lysosomes and is widely present in tissues, body fluids, and cells. It is most abundant in the prostate and renal proximal tubular cells. Changes in NAG activity are closely related to certain pathological states.

NAG degrades β-N-acetylaminoglucoside to produce p-nitrophenol, which has a maximum absorption peak at 400 nm. NAG activity is calculated by measuring the increase in absorbance.

Package List

Quality Standards and Safety Information

Transportation and Storage

Instructions

1. Preparation of Crude Enzyme Extract

1.1 Bacteria or Cultured Cells

1. Collect bacteria or cells into a centrifuge tube, centrifuge, and discard the supernatant.
2. Add Extraction Solution according to the ratio of bacteria or cells number (10⁴cells) to Extraction Solution volume (mL) = 500–1000:1. Recommended: add 500 × 10⁴bacteria or cells to 1 mL Extraction Solution.
3. Disrupt the bacteria or cells by ultrasonication in an ice bath at 20% power or 200 W: sonicate for 3 s, pause for 10 s, and repeat 30 times.
4. Centrifuge at 15000g and 4°C for 10 min.
5. Collect the supernatant and keep it on ice for testing.

1.2 Tissue

1. Add Extraction Solution according to the ratio of tissue mass (g) to Extraction Solution volume (mL) = 1:5–10. Recommended: weigh approximately 0.1 g tissue and add 1 mL Extraction Solution.
2. Homogenize in an ice bath.
3. Centrifuge at 15000g and 4°C for 10 min.
4. Collect the supernatant and keep it on ice for testing.

2. Reagent Preparation

Before use, add 2.5 mL distilled water to each bottle of Reagent I and dissolve thoroughly. Store unused Reagent I at -20°C.

3. Assay Procedure

1. Preheat the spectrophotometer for at least 30 min.
2. Set the wavelength to 400 nm and zero with distilled water.
3. Prepare the assay and control tubes as shown below.

Mix quickly and incubate in a 37°C precision water bath for 30 min.

Mix thoroughly and measure the absorbance at 400 nm. Calculate ΔA = A_measured- A_control. Each assay tube must have one corresponding control tube.

NAG Activity Calculation

The regression equation measured under standard conditions is y = 0.00543x + 0.0083, where x is the standard concentration (nmol/mL) and y is the absorbance value.

1. Serum or Plasma NAG Activity

Unit definition: the production of 1 nmol p-nitrophenol per minute in each mL of serum or plasma is defined as one unit of enzyme activity.

NAG activity (nmol/min/mL) = [(ΔA - 0.0083) ÷ 0.00543 × V_{total reaction}] ÷ V_sample÷ T = 61.39 × (ΔA - 0.0083)

2. Calculation Based on Sample Protein Concentration

Unit definition: the production of 1 nmol p-nitrophenol per minute in each mg of tissue protein is defined as one enzyme activity unit.

NAG activity (nmol/min/mg protein) = [(ΔA - 0.0083) ÷ 0.00543 × V_{total reaction}] ÷ (V_sample× Cpr) ÷ T = 61.39 × (ΔA - 0.0083) ÷ Cpr

3. Calculation Based on Sample Fresh Weight

Unit definition: the production of 1 nmol p-nitrophenol per minute in each g of tissue is defined as one enzyme activity unit.

NAG activity (nmol/min/g fresh weight) = [(ΔA - 0.0083) ÷ 0.00543 × V_{total reaction}] ÷ (W × V_sample÷ V_{total sample}) ÷ T = 61.39 × (ΔA - 0.0083) ÷ W

4. Calculation Based on Bacterial or Cell Density

Unit definition: the production of 1 nmol p-nitrophenol per minute by each 10,000 bacteria or cells is defined as one enzyme activity unit.

NAG activity (nmol/min/10⁴cells) = [(ΔA - 0.0083) ÷ 0.00543 × V_{total reaction}] ÷ (500 × V_sample÷ V_{total sample}) ÷ T = 0.123 × (ΔA - 0.0083)

Formula Parameters

Precautions

The 100T kit can test 48 samples.