Glucose-6-Phosphate Dehydrogenase Activity Colorimetric Assay Kit Kit
- Glucose-6-Phosphate Dehydrogenase (G6PD)
- Various Species
- Detection Method
- Activity Assay (AcA)
- Sample Type
- Cell Culture Supernatant, Plasma, Serum, Tissue Samples
- Glucose-6-phosphate dehydrogenase Assay Kit is a simple, sensitive and rapid assay detects the activity of G6PDH in a variety of samples. In the assay, glucose-6-phosphate is oxidized with the generation of a product which is utilized to convert a nearly colorless probe to an intensely colored product with an absorbance at 450nm. The G6PDH Assay Kit can detect as low as 0.04 mU G6PDH per well.
- Glucose-6-Phosphate Dehydrogenase (G6PDH) Activity Assay Kit: Colorimetric Assay for Measuring G6PDH Activity in Tissues, Erythrocytes etc. Rapid, Simple & Sensitive.
G6PDH Assay Buffer
G6PDH Positive Control
NADH Standard (0.5 μmol)
- Application Notes
- Measurement of G6PDH activity in various tissues and cells - Evaluation of pentose phosphate pathway
Further details regarding sample type: Cell and tissue culture supernatants, urine, plasma and serum, as well as many other biological fluids, etc.
1. Tissue or erythrocyte Sample Preparation: Samples (10-100 mg) should be rapidly homogenized with an equivalent volume of ice cold PBS or other buffer (pH 6.5-8). Add 1-50 µL samples into duplicate wells of a 96-well plate and bring volume to 50 µL with Assay Buffer. We suggest testing several doses of your samples to ensure readings are within the linear range.
2. Dilute Positive Control: Take 10 µL of the Positive Control and add 990 µL Assay Buffer. This should be a suitable dilution to get 0.1-1.0 OD in 30 minutes of incubation. Use 1-10 µL of the diluted Positive Control, adjust final volume to 50 µL with Assay Buffer.
3. Develop: Mix enough reagent for the number of samples and standards to be performed: For each well, prepare a total 50 µL Reaction Mix containing: Reaction Mix G6PDH Assay Buffer 46 µL G6PDH Substrate 2 µL G6PDH Developer 2 µL Add 50 µL of the Reaction Mix to each well containing the Positive Control or test samples. Measure O.D. 450 nm at T 1 to read A 1, measure O.D. 450 nm again at T 2 after incubating the reaction at 37 °C for 30 min (or longer if the G6PDH activity is low) to read A 2, protect from light. deltaA450 nm = A 2 - A 1. Note: It is essential to read A 1 and A 2 in the reaction linear range. It will be more accurate if you read the reaction kinetics. Then choose A 1, A 2, in the reaction linear range.
4. NADH Standard Curve: Add 0, 2, 4, 6, 8, and 10 µL of the 1.20 mM NADH Standard into 96-well plate in duplicate to generate 0, 2.5, 5.0, 7.5, 10.0, and 12.5nM/well standard. Bring the final volume to 50 µL with Assay Buffer, and then add 50 µL Reaction Mix to each standard, mix well. Measure at O.D.450 nm.
- Calculation of Results
Calculation: Subtract the background, plot NADH standard Curve. Apply the deltaA 450nm to the standard curve to get B (the NADH amount that was generated between T 1 and T 2 ). G6PDH Activity = Sample dilution = nM/min/mL = mU/mL Where: B is the NADH amount that was generated between T 1 and T 2 (in nmol). T 1 is the time of first reading (A 1 ) (in min). T 2 is the time of second reading (A 2 ) (in min). V is the pretreated sample volume added into the reaction well (in ml). One unit defines as the amount of enzyme that catalyzes the conversion of 1.0 µM of glucose-6-phosphate into 6-phosphoglucono-delta-lactone and generates 1.0 µM of NAD + to NADH per minute at 37 °C.
- For Research Use only
- -20 °C
- Expiry Date
- 12 months
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Vázquez-Medina, Zenteno-Savín, Forman, Crocker, Ortiz: "Prolonged fasting increases glutathione biosynthesis in postweaned northern elephant seals." in: The Journal of experimental biology, Vol. 214, Issue Pt 8, pp. 1294-9, 2011 (PubMed).
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- Glucose-6-Phosphate Dehydrogenase (G6PD)
- Alternative Name
- Glucose-6-Phosphate Dehydrogenase (G6PD ELISA Kit Abstract)
- G6PD1, g6pd, G6pd, G6pdx, g6pdh, g6pd2, fj78b06, wu:fj78b06, si:dkey-90a13.8, BA3433, G28A, G6PD, Gpdx, glucose-6-phosphate dehydrogenase, glucose-6-phosphate 1-dehydrogenase, glucose-6-phosphate 1-dehydrogenase (G6PD), glucose-6-P dehydrogenase, glucose-6-phosphate dehydrogenase L homeolog, glucose-6-phosphate 1-dehydrogenase Zwf, hexose-6-phosphate dehydrogenase/glucose 1-dehydrogenase, glucose-6-phosphate dehydrogenase X-linked, G6PD, g6pd, g6pD, zwf, LOC100120232, LACBIDRAFT_188936, G6pd, g6pd.L, CNG03280, Tb10.70.5200, H6PD, G6pdx
- Glucose-6-phosphate dehydrogenase (G6PDH) is a cytosolic enzyme in the pentose phosphate pathway, a metabolic pathway that supplies reducing energy to cells (such as erythrocytes) by maintaining the level of the co-enzyme nicotinamide adenine dinucleotide phosphate (NADPH). The NADPH in turn maintains the level of glutathione in these cells that helps protect the red blood cells against oxidative damage. Of greater quantitative importance is the production of NADPH for tissues actively engaged in biosynthesis of fatty acids and/or isoprenoids, such as the liver, mammary glands, adipose tissue, and the adrenal glands.