Glutamate Synthase (GOGAT) is a key enzyme in nitrogen assimilation and cycling within plants. It catalyzes the formation of glutamate from glutamine and α-ketoglutarate. Together with Glutamine Synthetase (GS), it forms the GS/GOGAT cycle, which participates in primary nitrogen uptake, the reabsorption of ammonia released during photorespiration, as well as ammonia assimilation and nitrogen fixation. GOGAT is primarily found in prokaryotes, yeast, and the proplastids of non-green tissues in higher plants.
Detection Principle: GOGAT uses NADH as an electron donor to catalyze the transfer of the amino group from glutamine to α-ketoglutarate, forming two molecules of glutamate. The rate of decrease in NADH absorbance at 340 nm reflects the level of GOGAT activity.
G1505937
| Component
| 48T
| 96T
| Storage
|
G1505937A
| Reagent Ⅰ
| 60 mL
| 120 mL
| 2-8℃
|
G1505937B
| Reagent Ⅱ
| 12 mL | 24 mL | 2-8℃
|
G1505937C
| Reagent Ⅲ
| 1EA | 2EA
| 2-8℃. Store in the dark. |
Note: Before formal testing, it is recommended to perform a preliminary experiment using 2-3 samples expected to have significant differences.
Required Instruments and Reagents
1. Microplate reader or UV spectrophotometer (capable of measuring absorbance at 340 nm)
2. 96-well UV plate or micro quartz cuvettes, adjustable pipettes and tips
3. Ice maker, refrigerated centrifuge
4. Deionized water
5. Homogenizer (for tissue samples)
Experimental Procedure
1. Reagent Preparation
Reagent Name
| Reagent Preparation
| Notes
|
Reagent Ⅰ
| Ready-to-use; Equilibrate to room temperature before use.
| Store at 4℃
|
Working Solution
| Prepare immediately before use: Take one bottle of Reagent Ⅲ, add 10.8 mL Reagent Ⅱ to it, and mix thoroughly.
| Prepare fresh before use; use on the same day. |
2. Sample Preparation
Notes:
(1) Fresh samples are recommended. During the assay, keep samples and all reagents on ice to prevent denaturation and inactivation.
(2) If protein concentration measurement is required, the use of Aladdin's B665595 BCA Protein Quantification Kit or R1491648 Ready-to-Use BCA Protein Quantification Kit is recommended. Since Reagent Ⅰ contains a certain concentration of protein (approximately 10 mg/mL), the intrinsic protein content of Reagent Ⅰ must be subtracted when determining the sample protein concentration.
2.1 Tissue
Weigh approximately 0.1 g of sample, add 1 mL of Reagent Ⅰ, and homogenize on ice. Centrifuge at 8,000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for testing.
2.2 Cells (Bacteria)
Collect 5 million cells (bacteria) into a centrifuge tube. Wash with cold PBS, centrifuge, and discard the supernatant. Add 1 mL of Reagent Ⅰ. Disrupt the cells using ice-bath ultrasonication for 5 minutes (power 20% or 200 W, ultrasonic for 3 s, interval 7 s, repeat 30 times). Then centrifuge at 8,000 g, 4°C for 10 min. Collect the supernatant and keep it on ice for testing.
2.3 Serum (Plasma) samples
Can be detected directly.
3. Experimental Steps
3.1 Preheat the microplate reader or UV spectrophotometer for at least 30 minutes. Set the wavelength to 340 nm. For the UV spectrophotometer, zero it with deionized water.
3.2 Incubate the Working Solution at 37°C (for mammals) or 25°C (for other species) for 30 minutes.
3.3 Operation Table (Perform the following steps in a 96-well UV plate or micro quartz cuvette)
Reagent
| Test Well (μL)
|
Sample
| 20
|
Working Solution
| 180
|
3.4 Mix quickly and measure the absorbance at 340 nm immediately. Record the absorbance values at 20 seconds and 5 minutes 20 seconds, denoted as A1 and A2 respectively. Calculate ΔATest = A1 - A2.
Note: It is recommended to perform a preliminary experiment with 2-3 samples expected to have large differences before the formal experiment. If ΔATest is greater than 0.5, the sample can be further diluted with Reagent Ⅰ, and the calculated result should be multiplied by the dilution factor. Since enzyme activity is calculated based on the absorbance change per unit time, it is not recommended to measure multiple samples simultaneously.
4. Calculation of Results
Note: We provide two types of calculation formulas: the derived process formula and the simplified formula. They are completely equivalent. It is recommended to use the simplified formula in bold as the final calculation formula.
4.1 Calculation Formulas for using 96-well UV Plate
(1) Calculation of GOGAT Activity in Serum (Plasma)
Unit Definition: One unit of enzyme activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per milliliter of serum (plasma).
GOGAT (U/mL) = [ΔATest × VReaction Total ÷ (ε × d) × 109] ÷ VSample ÷ T = 643 × ΔATest
(2) Calculation of GOGAT Activity in Tissue
A. Calculation based on sample protein concentration
Unit Definition: One unit of enzyme activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per milligram of tissue protein.
GOGAT (U/mg prot) = [ΔATest × VReaction Total ÷ (ε × d) × 109] ÷ (VSample × Cpr) ÷ T = 643 × ΔATest ÷ Cpr
B. Calculation based on sample fresh weight
Unit Definition: One unit of enzyme activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per gram of tissue.
GOGAT (U/g fresh weight) = [ΔATest × VReaction Total ÷ (ε × d) × 109] ÷ (W ÷ VReagent Ⅰ × VSample) ÷ T = 643 × ΔATest ÷ W
C. Calculation based on cell (bacterial) density
Unit Definition: One unit of enzyme activity is defined as the amount of enzyme that consumes 1 nmol of NADH per minute per 10⁴ cells (bacteria).
GOGAT (U/10⁴) = [ΔATest × VReaction Total ÷ (ε × d) × 109] ÷ (VSample ÷ VReagent Ⅰ × 500) ÷ T = 1.286 × ΔATest
Parameter Description:
VReaction Total: Total reaction system volume, 0.2 mL = 2 × 10-4 L;
VReagent Ⅰ: Volume of Reagent Ⅰ added, 1 mL;
VSample: Volume of sample added, 0.02 mL;
ε: Molar extinction coefficient of NADH, 6.22 × 10³ L/mol/cm;
d: Light path for 96-well UV plate, 0.5 cm;
Cpr: Protein concentration (mg/mL);
T: Reaction time, 5 min;
W: Sample mass, g;
500: Total number of cells (bacteria), 5 million;
109: Unit conversion factor, 1 mol = 10⁹ nmol.
4.2 Calculation Formulas for using Micro Quartz Cuvette
Adjust the light path d from 0.5 cm to 1 cm in the formulas above for calculation.
Precautions
1. Before formal testing, it is recommended to perform a preliminary experiment using 2-3 samples expected to have significant differences.
2. This product is for scientific research use only. It is not intended for clinical diagnosis. For your safety and health, please wear lab coats and disposable gloves during operation.
