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

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Overview SDS Protocol

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 G6PDH predispose individuals to non-immune hemolytic anemia. AAT Bioquest's Amplite® Colorimetric Glucose-6-Phosphate Dehydrogenase Assay Kit provides a simple, sensitive and rapid absorbance-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 chromogenic NADPH sensor. The absorption signal can be read by an absorption microplate reader at an absorbance ratio of Abs_{575 nm} to Abs_{605 nm}. With the G6PD assay kit, we were able to detect as little as 3 mU/ml G6PD in a 100 µL reaction volume.

Platform

Absorbance microplate reader

Absorbance	575/605 nm
Recommended plate	Clear bottom

Components

Example protocol

AT A GLANCE

Protocol summary

Prepare G6PD working solution (50 µL)
Add G6PD standards or test samples (50 µL)
Incubate at room temperature for 30 minutes - 2 hours
Monitor absorbance ratio increase at A_575nm/A_605nm

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

PREPARATION OF STOCK SOLUTION

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

G6PD standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13807

Add 10 µL of 100 U/mL G6PD standard solution into 990 µL 1x PBS buffer to generate 1000 mU/mL G6PD standard solution. Take 1000 mU/mL G6PD standard solution and perform 1:3 serial dilutions in 1x PBS buffer to get serially diluted G6PD standards (G6PD7 - G6PD1). Note: Diluted G6PD standard solution is unstable and should be used within 4 hours.

PREPARATION OF WORKING SOLUTION

1. Add 5 mL of Assay Buffer (Component B) into one bottle of Enzyme Probe (Component A), and mix well.

2. Add 50 µL of 100X NADP stock solution into the bottle of Component A+B and mix well to make G6PD working solution. Note: This G6PD working solution is enough for one 96-well plate. It is unstable at room temperature and should be used promptly within 2 hours. Avoid exposure to light.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of G6PD standards and test samples in a white clear bottom 96-well microplate. G6PD=D-Glucose-6-Phosphate Dehydrogenase Standards (G6PD1 - G6PD7, 0.4 to 300 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 Dilutions (0.4 to 300 mU/mL)
BL	50 µL	Dilution Buffer
TS	50 µL	test sample

Prepare G6PD standards (G6PD), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 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 G6PD assay volume of 100 µL/well. For a 384-well plate, add 25 µL of G6PD 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 absorbance ratio increase with an absorbance plate reader at A_575nm/A_605nm.

Images

Figure 1. G6PD dose response was measured with Amplite® Colorimetric Glucose-6-Phosphate Dehydrogenase Assay Kit in a white clear bottom plate using a SpectraMax Plus (Molecular Devices) microplate reader.

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

Effects of glucose-6-phosphate dehydrogenase deficiency on the metabolic and cardiac responses to obesogenic or high-fructose diets
Authors: Hecker PA, Mapanga RF, Kimar CP, Ribeiro RF, Jr., Brown BH, O'Connell KA, Cox JW, Shekar KC, Asemu G, Essop MF, Stanley WC.
Journal: Am J Physiol Endocrinol Metab (2012): E959

Glucose 6 phosphate dehydrogenase deficiency: a case series
Authors: E, undefined and i Eberle S, Garcia Rosolen N, Urtasun C, Sciuccati G, Diaz L, Savietto V, C and as A, Avalos Gomez V, Cervio C, Bonduel M, Feliu Torres A.
Journal: Arch Argent Pediatr (2011): 354

Glutathione-dependent enzymes and glucose-6-phosphate dehydrogenase of blood in patients with lymphosarcoma (non-Hodgkin's disease)
Authors: Gavriliuk LA, Korchmaru IF, Robu MV, Lysyi LT.
Journal: Biomed Khim (2011): 225

Glucose-6-phosphate dehydrogenase status and severity of malarial anaemia in Nigerian children
Authors: Orimadegun AE, Sodeinde O.
Journal: J Infect Dev Ctries (2011): 792

Implementation and analysis of a pilot in-hospital newborn screening program for glucose-6-phosphate dehydrogenase deficiency in the United States
Authors: Nock ML, Johnson EM, Krugman RR, Di Fiore JM, Fitzgerald S, S and haus LM, Walsh MC.
Journal: J Perinatol (2011): 112