Amplite® Fluorimetric Glucose-6-Phosphate Dehydrogenase (G6PD) Assay Kit

Name: Amplite® Fluorimetric Glucose-6-Phosphate Dehydrogenase (G6PD) Assay Kit
Brand: AAT Bioquest
SKU: 13806
Price: 341 USD
Availability: InStock

Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the conversion of glucose-6-phosphate to 6-phosphoglucono-δ-lactone, the first and rate-limiting step in the pentose phosphate pathway. It is critical metabolic pathway that supplies reducing energy to cells (such as erythrocytes) by maintaining the level of co-enzyme nicotinamide adenine dinucleotide phosphate (NADPH), and for the production of pentose sugars. The production of NADPH is of great importance for tissues actively engaged in biosynthesis of fatty acids and/or isoprenoids, such as the liver, mammary glands, adipose tissue, and the adrenal glands. The NADPH also maintains the level of glutathione in these cells that helps protect the red blood cells against oxidative damage. Deficiencies in G6PD predispose individuals to non-immune hemolytic anemia. AAT Bioquest's Amplite® Fluorimetric Glucose-6-Phosphate Dehydrogenase Assay Kit provides a simple, sensitive and rapid fluorescence-based method for detecting G6PD in biological samples such as serum, plasma, urine, as well as in cell culture samples. In the enzyme coupled assay, G6PD activity is proportionally related to the concentration of NADPH that is specifically monitored by a fluorogenic NADPH sensor to yield a highly red fluorescence product. The fluorescence signal can be read with a fluorescence microplate reader. With the G6PD assay kit, we were able to detect as little as 1 mU/ml G6PD in a 100 µL reaction volume.

Example protocol

AT A GLANCE

Protocol Summary

Prepare G6PD standards or test samples (50 µL)
Add G6PD working solution (50 µL)
Incubate at RT for 30 minutes - 2 hours
Monitor fluorescence increase at Ex/Em = 540/590 nm

Important Thaw each kit component at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. NADP stock solution (100X)

Add 100 µL of H₂O into the vial of NADP (Component C) to make 100X NADP stock solution.

2. G6PD standard solution (100 U/mL)

Add 100 µL of H₂O or 1X PBS buffer into the vial of G6PD Standard (Component D) to make 100 U/mL G6PD standard solution.

PREPARATION OF STANDARD SOLUTION

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/13806

G6PD standard

Add 10 µL of G6PD standard solution into 990 µL 1X PBS buffer to generate 1000 mU/mL G6PD standard solution. Take 15 µL of 1000 mU/mL G6PD standard solution into 485 uL of 1X PBS buffer to generate 30 mU/mL G6PD (G6PD7), and then perform 1:3 serial dilutions to get serial dilutions of G6PD standard (G6PD6 - G6PD1). Note: Diluted G6PD standard solution is unstable, and should be used within 4 hours.

PREPARATION OF WORKING SOLUTION

Add 5 mL of Assay Buffer (Component B) into one bottle of Enzyme Probe (Component A). Add 50 µL NADP stock solution (100X) into the bottle of Component A, and mix well.
Note This G6PD assay working solution is enough for one 96-well plate.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of G6PD standards and test samples in a solid black 96-well microplate. G6PD = D-Glucose-6-Phosphate Dehydrogenase standard (G6PD1 - G6PD7, 0.04 to 30 mU/mL); BL = blank control; TS = test samples.

BL	BL	TS	TS
G6PD1	G6PD1	...	...
G6PD2	G6PD2	...	...
G6PD3	G6PD3
G6PD4	G6PD4
G6PD5	G6PD5
G6PD6	G6PD6
G6PD7	G6PD7

Table 2. Reagent Composition for each well.

Well	Volume	Reagent
G6PD1 - G6PD7	50 µL	Serial Dilution (0.04 to 30 mU/mL)
BL	50 µL	Dilution Buffer (PBS)
TS	50 µL	Test Sample

Prepare G6PD standards (G6PD), blank controls (BL), and test samples (TS) according to the layout provided in Table 1 and Table 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
Add 50 µL of G6PD working solution to each well of G6PD standard, blank control, and test samples to make the total assay volume of 100 µL/well. For a 384-well plate, add 25 µL of working solution into each well instead, for a total volume of 50 µL/well.
Incubate the reaction at room temperature for 30 minutes to 2 hours, protected from light.
Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 540/590 nm, cut off at 570 nm.

Citations

View all 1 citations: Citation Explorer

A novel pathogenic variant in the glucokinase gene found in two Japanese siblings with maturity-onset diabetes of the young 2
Authors: Tanaka, Satoshi and Akagawa, Hiroyuki and Azuma, Kenkou and Watanabe, Kaoru and Higuchi, Sayaka and Iwasaki, Naoko
Journal: Endocrine Journal (2023): EJ22--0541

References

View all 71 references: Citation Explorer

The microsomal enzyme 17beta-hydroxysteroid dehydrogenase 3 faces the cytoplasm and uses NADPH generated by glucose-6-phosphate dehydrogenase
Authors: Legeza B, Balazs Z, Nashev LG, Odermatt A.
Journal: Endocrinology (2013): 205

Molecular characterization of glucose-6-phosphate dehydrogenase deficient variants in Baghdad city - Iraq
Authors: Al-Musawi BM, Al-Allawi N, Abdul-Majeed BA, Eissa AA, Jubrael JM, Hamamy H.
Journal: BMC Blood Disord (2012): 4

High prevalence of hemoglobin disorders and glucose-6-phosphate dehydrogenase (G6PD) deficiency in the Republic of Guinea (West Africa)
Authors: Millimono TS, Loua KM, Rath SL, Relvas L, Bento C, Diakite M, Jarvis M, Daries N, Ribeiro LM, Manco L, Kaeda JS.
Journal: Hemoglobin (2012): 25

Effects of laparoscopic Roux-en-Y gastric bypass on glucose-6 phosphate dehydrogenase activity in obese type 2 diabetics
Authors: Schneider AM, Rawat D, Weinstein LS, Gupte SA, Richards WO.
Journal: Surg Endosc (2012): 823

A novel cytofluorometric assay for the detection and quantification of glucose-6-phosphate dehydrogenase deficiency
Authors: Shah SS, Diakite SA, Traore K, Diakite M, Kwiatkowski DP, Rockett KA, Wellems TE, Fairhurst RM.
Journal: Sci Rep (2012): 299

Page updated on November 21, 2024

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