Microassay of Glucose Oxidase (GOD) Test Kit

Glucose oxidase (GOD) test kit
Micro method
Item No.BC0695
Specifications100T/96S

Product Content:
Extraction solution:Liquid 100mL x 1 bottle, stored at 4 ℃;
Reagent 1:Liquid 15mL x 1 bottle, stored at 4 ℃;
Reagent 2:Liquid 3mL x 1 bottle, stored at 4 ℃;
Reagent 3:Liquid 1mL x 1 tube, stored at -20 ℃, can be packaged and stored after melting.

Product Description:
GOD (EC 1.1.3.4) is widely present in animals and plants, catalyzing the oxidation of glucose to produce gluconic acid and generate H2O2, which is one of the metabolic pathways for the production of reactive oxygen species in organisms.
GOD catalyzes the production of H 2 O 2 from glucose, and peroxidase catalyzes the decomposition of H 2 O 2 in the presence of oxygen, which in turn oxidizes ortho anisidine to produce colored substances. The color intensity is linearly related to the activity of glucose oxidase.

Self provided equipment and instruments are required
Visible spectrophotometer/microplate reader, water bath, desktop centrifuge, adjustable pipette, microcalorimeter/96 well plate, mortar, ice and distilled water

operating steps
1、 Preparation for Sample Measurement
1. Organizational processing: Weigh about 0.1g of tissue and add 1mL of extraction solution for ice bath homogenization; Centrifuge 8000g at 4 ℃ for 10 minutes, take the supernatant, and place it on ice for testing.
2. Serum (plasma): direct detection.
2、 Measurement steps
1. Preheat the spectrophotometer or enzyme-linked immunosorbent assay (ELISA) reader for at least 30 minutes, adjust the wavelength to 500nm, and zero the distilled water.
2. Preparation of GOD working solution: Mix 15mL of reagent one with 3mL of reagent two, and adjust the pH to 5.1 (ready to use).
3. Sample addition table:
Reagent Name (μ L) Determination Tube
GOD working fluid 174
Reagent 3 6
Mix well, water bath at 37 ℃ (mammals) or 25 ℃ (other species) for 5 minutes
Sample 20
Before sample determination, put the GOD working solution and reagent three in a 37 ℃ water bath for insulation, and then add the above reagents in the order of trace ratio
Start timing while adding samples to the color dish/96 well plate; Record the initial absorbance A1 at a wavelength of 500 nm for 20 seconds and the absorbance A2 at 1 minute and 20 seconds. Calculate Δ A=A2-A1.
3、 Calculation of GOD vitality units
a. The calculation formula for measuring with a trace quartz colorimetric dish is as follows
1. Calculation of GOD vitality in animal tissues
(1) Calculated by protein concentration
The definition of the unit: One enzyme activity unit is defined as the catalytic production of 1 μ mol of oxidized ortho phenylenediamine per minute per mg of tissue protein in the reaction system.
GOD (U/mg prot)=Δ A ÷ (ε× d) × V inverse total ÷ (Cpr × V sample) ÷ T=1333 × Δ A ÷ Cpr
(2) Calculated based on the fresh weight of the sample
The definition of a unit: One enzyme activity unit is defined as the catalytic production of 1 μ mol of oxidized ortho phenylenediamine per minute per g of tissue in the reaction system.
GOD (U/g fresh weight)=Δ A ÷ (ε× d) × V inverse total ÷ (W ÷ V extraction × V sample) ÷ T=1333 × Δ A ÷ W
2. Calculation of serum (plasma) GOD activity
The definition of the unit: One enzyme activity unit is defined as the catalytic production of 1 μ mol of oxidized ortho phenylenediamine per minute per mL of serum (slurry) in the reaction system.
GOD (U/mL)=Δ A ÷ d ÷ ε× V inverse total ÷ T ÷ V sample=1333 × Δ A
V anti total: total reaction volume, 0.2mL; V sample: added sample volume, 0.02mL; V extraction, added extraction solution volume,
1mL; CPR: sample protein concentration, mg/mL； W: Sample fresh weight, g; d: optical path, 1cm; ε: extinction coefficient of oxidized ortho fennel amine, 7.5 × 10-3 mL/μ mol/cm; T: Reaction time, 1 minute.
b. The calculation formula determined using a 96 well plate is as follows
1. Calculation of GOD vitality in animal tissues
(1) Calculated by protein concentration
The definition of the unit: One enzyme activity unit is defined as the catalytic production of 1 μ mol of oxidized ortho phenylenediamine per minute per mg of tissue protein in the reaction system.
GOD (U/mg prot)=Δ A ÷ (ε× d) × V inverse total ÷ (Cpr × V sample) ÷ T=2222 × Δ A ÷ Cpr
(2) Calculated based on the fresh weight of the sample
The definition of a unit: One enzyme activity unit is defined as the catalytic production of 1 μ mol of oxidized ortho phenylenediamine per minute per g of tissue in the reaction system.
GOD (U/g fresh weight)=Δ A ÷ (ε× d) × V inverse total ÷ (W ÷ V extraction × V sample) ÷ T=2222 × Δ A ÷ W
2. Calculation of serum (plasma) GOD activity
The definition of the unit: One enzyme activity unit is defined as the catalytic production of 1 μ mol of oxidized ortho phenylenediamine per minute per mL of serum (slurry) in the reaction system.
GOD (U/mL)=Δ A ÷ d ÷ ε× V inverse total ÷ T ÷ V sample=2222 × Δ A
V anti total: total reaction volume, 0.2mL; V sample: added sample volume, 0.02mL; CPR: sample protein concentration, mg/mL；
W: Sample fresh weight, g; d: optical path, 0.6cm; ε: extinction coefficient of oxidized ortho fennel amine, 7.5 × 10-3 mL/μ mol/cm; T: Reaction time, 1 minute.
1. The activity of GOD varies in different homogenized tissues. Before conducting formal experiments, please conduct 1-2 pre tests. If A2-A1>1,
This indicates that the tissue vitality is too high and must be diluted with extraction solution to obtain a suitable concentration of homogenate supernatant, so that A2-A1<1,
To improve detection sensitivity.
If A1>A2 phenomenon occurs during the experiment, please dilute the sample with extraction solution to an appropriate concentration
2. Two people are doing this experiment simultaneously, one comparing colors and the other timing, to ensure the accuracy of the experimental results.
3. This kit is only applicable to animal tissues and animal serum (slurry).

Microassay of Glucose Oxidase (GOD) Test Kit Microassay of Glucose Oxidase (GOD) Test Kit