NB-64-97472-50T | Glucose Assay Kit (Spectrophotometry) Neobiotech

Detection Principle

Glucose oxidase catalyzes the oxidation of glucose to gluconic acid, producing hydrogen peroxide. Peroxidase then catalyzes the oxidation of 4-aminoantipyrine coupled with phenol by hydrogen peroxide to form a colored compound, which has a characteristic absorption peak at 505 nm. The absorbance intensity is directly proportional to the original glucose concentration.

Packing

Taking 50T/48S packing for example:

Components	Packing	Storage
CB0082S-A	Glucose Solution (2 μmol/mL), 10 mL per vial	4 ℃
CB0082S-B	Liquid, 25 mL per vial	4 ℃
CB0082S-C	Liquid, 25 mL per vial	4 ℃
Before use, mix CB0082S-B and CB0082S-C at a 1:1 ratio (equal volumes). Prepare only the amount required for immediate use.

Before the formal assay, be sure to select 2–3 samples with relatively large expected differences for a preliminary test.

Instructions

I.Required Materials (to be prepared by the user)

Visible spectrophotometer, 1 mL glass cuvette, water bath, analytical balance, centrifuge, adjustable micropipettes, distilled water.

II.Glucose Extraction

1. Tissue samples

Homogenize the tissue with distilled water at a ratio of tissue weight (g) : distilled water volume (mL) = 1 : 5–10
(recommended: weigh ~0.1 g tissue and add 1 mL distilled water).

Boil the homogenate in a boiling water bath for 10 minutes (cap tightly to prevent water loss).

After cooling, centrifuge at 8000 × g for 10 minutes at room temperature.

Collect the supernatant for analysis.

2. Bacterial or cell samples

Collect bacteria or cells into centrifuge tubes and centrifuge; discard the supernatant.

Add distilled water according to the ratio of bacterial or cell number (10⁴ cells) : distilled water volume (mL) = 500~1000 : 1 (recommended: add 1 mL distilled water to 5 × 10⁶ bacteria or cells).

Disrupt bacteria or cells by ultrasonication (ice bath, 20% power or 200 W; sonicate for 3 s with 10 s intervals, repeat 30 times).

Boil in a boiling water bath for 10 minutes (cap tightly to prevent water loss).

After cooling, centrifuge at 8000 × g for 10 minutes at 25 °C.

Collect the supernatant for analysis.

3. Serum (plasma): Measure directly.

III.Assay Procedure:

1.Preheat the spectrophotometer for at least 30 minutes, set the wavelength to 505 nm, and zero with distilled water.

2.Preparation of working solution: Mix equal volumes of CB0082S-B and CB0082S-C before use; prepare only the amount needed.

3.Add the following reagents into a 1.5 mL centrifuge tube:

	Blank Tube（μL）	Control Tube（μL）	Sample Tube（μL）
Sample			100
CB0082S-A		100
Distilled Water	100
Working Solution	900	900	900
Mix well and incubate in a water bath at 37 °C (for mammals) or 25 °C (for other species) for 15 minutes. Measure the absorbance A at 505 nm. Record the absorbance of the blank, standard, and test tubes as A1, A2, and A3, respectively. Note: Only one tube is needed for the blank and the standard.

IV.Calculation of Glucose / Blood Glucose Content:

1.Based on sample protein concentration

Glucose content (μmol/mg protein)== (Cstd×V1)×(A3-A1)÷(A2-A1)÷(V1×Cpr)=2×(A3-A1)÷(A2-A1)÷Cpr

2.Based on sample mass

Glucose content (μmol/g fresh weight)= (Cstd×V1)×(A3-A1)÷(A2-A1)÷(W×V1÷V2)=2×(A3-A1)÷(A2-A1)÷W

3.Based on bacterial or cell density

Glucose content (μmol/10⁴ cells)=(Cstd×V1)×(A3-A1)÷(A2-A1)÷(500×V1÷V2) = 0.004×(A3-A1)÷(A2-A1)

4.Blood glucose content

Blood glucose (μmol/mL)=Cstd×(A3-A1)÷(A2-A1) = 2×(A3-A1)÷(A2-A1)

Note:

Cstd：2µmol/mL；

V1：Volume of sample added, 100µL=0.1mL；

V2：Volume of the sample extraction，1mL；

Cpr：Protein concentration，mg/mL；

W：Sample weight，g；

500：total number of cells or bacteria, (5 × 10⁶)

Precautions

1.The product is for R&D use only, not for diagnostic procedures, food, drug, household or other uses.

2.Please wear a lab coat and disposable gloves.

Glucose Assay Kit (Spectrophotometry)