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

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G6PD dose response was measured with Amplite™ Fluorimetric Glucose-6-Phosphate Dehydrogenase Assay Kit in a 96-well solid black plate using a Gemini (Molecular Devices) microplate reader.
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200 Tests 13806 $295


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Telephone: 1-800-990-8053
Fax: 1-408-733-1304
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Overview

Ex/Em (nm)571/585
Storage Freeze (<-15 °C)
Minimize light exposure
InstrumentsFluorescence microplate reader
Category Cell Biology
Cell Metabolism
Related Redox Enzymes
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 at Ex/Em = 540 nm/590 nm. With the G6PD assay kit, we were able to detect as little as 1 mU/ml G6PD in a 100 µL reaction volume.




Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.
At a glance

Protocol summary

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

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

Key parameters
Instrument:Fluorescence microplate reader
Excitation:540 nm
Emission:590 nm
Cutoff:570 nm
Recommended plate:Solid black
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
G6PD standard

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

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
  1. 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.

  2. 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.

  3. Incubate the reaction at room temperature for 30 minutes to 2 hours, protected from light.

  4. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 540/590 nm, cut off at 570 nm.
Example data analysis and figures

The reading (RFU) obtained from the blank standard well is used as a negative control. Subtract this value from the other standards' readings to obtain the base-line corrected values. Then, plot the standards' readings to obtain a standard curve and equation. This equation can be used to calculate Glucose-6-Phosphate Dehydrogenase samples. We recommend using the Online Linear Regression Calculator which can be found at:

https://www.aatbio.com/tools/linear-logarithmic-semi-log-regression-online-calculator/

Figure 1. G6PD dose response was measured with Amplite™ Fluorimetric Glucose-6-Phosphate Dehydrogenase Assay Kit in a 96-well solid black plate using a Gemini (Molecular Devices) microplate reader.

Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





References & Citations

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

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

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

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

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

Alternative targeting of Arabidopsis plastidic glucose-6-phosphate dehydrogenase G6PD1 involves cysteine-dependent interaction with G6PD4 in the cytosol
Authors: Meyer T, Holscher C, Schwoppe C, von Schaewen A.
Journal: Plant J (2011): 745

Frequency of glucose-6-phosphate dehydrogenase deficiency in malaria patients from six African countries enrolled in two randomized anti-malarial clinical trials
Authors: Carter N, Pamba A, Duparc S, Waitumbi JN.
Journal: Malar J (2011): 241

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, Sandhaus LM, Walsh MC.
Journal: J Perinatol (2011): 112


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Additional Documents

 
Safety Data Sheet (SDS)


Catalogs
1. Enzyme Probes & Assay Kits

Application Notes
1. AssayWise Letters 2013, Vol 2(1)

Certificate of Analysis