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Amplite® Fluorimetric Glutathione Peroxidase Assay Kit *Blue Fluorescence*

Glutathione Peroxidase (GPx) dose response was measured with Amplite® Fluorimetric Glutathione Peroxidase Assay Kit (Cat#11560) on a solid black 96-well plate using a Gemini microplate reader (Molecular Devices).
Glutathione Peroxidase (GPx) dose response was measured with Amplite® Fluorimetric Glutathione Peroxidase Assay Kit (Cat#11560) on a solid black 96-well plate using a Gemini microplate reader (Molecular Devices).
Glutathione Peroxidase (GPx) dose response was measured with Amplite® Fluorimetric Glutathione Peroxidase Assay Kit (Cat#11560) on a solid black 96-well plate using a Gemini microplate reader (Molecular Devices).
Activities of three antioxidant enzymes in host-derived structures surrounding developing Drino inconspicuoides larva. (a) Superoxidase dismutase (SOD) activity; (b) Glutathione peroxidase (GPx) activity; (c) Catalase activity. Each value is expressed as the mean ± standard deviation of samples (n = 5). Different letters above each bar denote significant differences (p < 0.01). GPx activity was detected using the Amplite Fluorimetric Glutathione Peroxidase Assay Kit (AAT Bioquest, CA, USA). Source: <b>Cloak Scavenges the Reactive Oxygen Species around the Larvae of Drino inconspicuoides (Diptera: Tachinidae)</b> by Zhang K, Nakamura S, Furukawa S. <em>Insects</em>. July 2023.
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OverviewpdfSDSpdfProtocol


Glutathione peroxidase is an enzyme family with peroxidase activity to protect the organism from oxidative damage. Glutathione peroxidase plays an important role in reducing organic hydroperoxides such as lipid hydroperoxides to their corresponding alcohols, or reducing free hydrogen peroxide to water. Glutathione peroxidase guards against oxidative damage to cell membranes and other oxidant-sensitive sites in cells. The altered glutathione peroxidase levels correlate with lesions caused by many common and complex diseases. Glutathione peroxidase level is measured in biological samples as a potential indicator for the potential treatment of cancer, diabetes, neurodegenerative and cardiovascular diseases. AAT Bioquest's Fluorimetric Glutathione Peroxidase Assay Kit offers a sensitive fluorimetric assay for measuring glutathione peroxidase levels in biological samples. This assay is based on the oxidation of glutathione (GSH) to oxidized glutathione (GSSG) catalyzed by glutathione peroxidase. The generated GSSG is recycled to its reduced state GSH by glutathione reductase (GR) and NADPH to generate NADP+ that can be specifically monitored using Quest Fluor™ NADP Probe, our newly developed proprietary NADP sensor. The NADP sensor reacts only with NADP to generate a fluorescent product. The fluorescence signal can be measured with a fluorescence microplate reader and is directly proportional to the glutathione peroxidase activity. Compared to other commercial kits that measure the decrease in absorbance of NADPH at 340 nm, our Quest Fluor™ NADP Probe can be used for quantify NADP level directly. With this fluorimetric glutathione peroxidase assay, as low as 1.25 mU/mL glutathione peroxidase can be detected in a 155 µL reaction volume.

Platform


Fluorescence microplate reader

Excitation420 nm
Emission480 nm
Cutoff430 nm
Recommended plateSolid black

Components


Example protocol


AT A GLANCE

Protocol Summary
  1. Prepare GPx standards or test samples (50 µL)
  2. Add GPx working solution (50 µL)
  3. Incubate at room temperature for 30 min
  4. Add 20 µL Quest Fluor™ NADP Probe
  5. Add 20 µL NADP Assay Solution
  6. Incubate at room temperature for 10 - 20 min
  7. Add 15 µL Enhancer Solution
  8. Incubate at room temperature for 30 - 60 min
  9. Record Fluorescence at Ex/Em= 420/480nm (Cutoff = 430 nm)
Important Note

To achieve the best results, it’s strongly recommended to use the black plates. Thaw one vial of 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

Glutathione Peroxidase (GPx) standard solution (10 U/mL)

Add 50 µL of ddH2O or 1× PBS buffer into the vial of GPx standard (Component A) to make 10 U/mL standard solution.

GSH stock solution (100X)

Add 100 µL of ddH2O into the vial of GSH (Component D) to make 100X GSH stock solution.

GPx Substrate stock solution (100X)

Add 100 µL of ddH2O into the vial of substrate (Component E) to make 100X GPx Substrate stock solution.

PREPARATION OF STANDARD SOLUTIONS

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/11560

GPx standard
Add 10 µL of GPx standard solution (10 U/mL) into 990 µL 1x PBS buffer + 0.1% BSA to generate 100 mU/mL GPx standard solution (GP7). Take 100 mU/mL GPx standard solution (GP7) and perform 1:1.5 serial dilutions in PBS + 0.1% BSA to get serial dilutions of GPx standard (GP6 - GP1). Note: Diluted GPx 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 a bottle of Enzyme Mix (Component C).

  2. Add 50 µL GSH stock solution (Component D), 50 µL GPx Substrate stock solution (Component E) into the bottle of Component B+C, and mix well to make GPx working solution (Component B+C+D+E). Note: This GPx working solution is enough for one 96-well plate. It is not stable; please use it promptly. It is not recommend storing unused GPx working solution.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of GPx standards and test samples in a solid black 96-well microplate. GP= GPx Standards (GP1 - GP7, 8.78 to 100 mU/mL), BL=Blank Control, TS=Test Samples.

BLBLTSTS
GP1GP1......
GP2GP2......
GP3GP3
GP4GP4
GP5GP5
GP6GP6
GP7GP7

Table 2. Reagent composition for each well.

WellVolumeReagent
GP1 - GP750 µL

Serial Dilution (8.78 to 100 mU/mL)

BL50 µL

1X PBS Buffer + 0.1% BSA

TS50 µLTest Sample
Run GPx assay:
  1. Prepare GPx standards (GP), 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.
  2. Add 50 µL of GPx working solution to each well of GPx standard, blank control, and test samples to make the total assay volume of 100 µL/well. For a 384-well plate, add 25 µL GPx working solution into each well instead, for total of 50 µL/well.
  3. Incubate the reaction at room temperature for 30 minutes, protected from light.
Run NADP assay:
  1. Add 20 µL Quest Fluor™ NADP Probe (Component F) into each well of GPx standard, blank control, and test samples, mix well.
  2. Add 20 µL NADP Assay Solution (Component G) into each well, mix well. For a 384-well plate, add 25 µL of sample and 10 µL of Quest Fluor™ NADP Probe (Component F) and 10 µL NADP Assay Solution (Component G) into each well.
  3. Incubate the reaction at room temperature for 10 - 20 minutes, protected from light.
  4. Add 15 µL Enhancer (Component H) to each well to make the total assay volume of 155 µL/well, and incubate at room temperature for 30 - 60 minutes, protected from light. For a 384-well plate, add 7.5 µL Enhancer.
  5. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 420/480 nm (Cutoff = 430nm).

Images


Citations


View all 2 citations: Citation Explorer
Cloak Scavenges the Reactive Oxygen Species around the Larvae of Drino inconspicuoides (Diptera: Tachinidae)
Authors: Zhang, Kai and Nakamura, Satoshi and Furukawa, Seiichi
Journal: Insects (2023): 602
Histidine-rich glycoprotein possesses antioxidant activity through self-oxidation and inhibition of hydroxyl radical production via chelating divalent metal ions in Fenton's reaction
Authors: Wake, Hidenori and Takahashi, Yohei and Yoshii, Yukinori and Gao, Shangze and Mori, Shuji and Wang, Dengli and Teshigawara, Kiyoshi and Nishibori, Masahiro
Journal: Free Radical Research (2020): 649--661

References


View all 52 references: Citation Explorer
Loss of glutathione peroxidase 7 promotes TNF-alpha-induced NF-kappaB activation in Barrett's carcinogenesis
Authors: Peng DF, Hu TL, Soutto M, Belkhiri A, El-Rifai W.
Journal: Carcinogenesis (2014): 1620
Oxygen Modulates the Glutathione Peroxidase Activity during the L6 Myoblast Early Differentiation Process
Authors: Hidalgo M, Marchant D, Quidu P, Youcef-Ali K, Richalet JP, Beaudry M, Besse S, Launay T.
Journal: Cell Physiol Biochem (2014): 67
Glutathione S-transferase, glutathione peroxidase and acetylcholinesterase activities in mussels transplanted to harbour areas
Authors: Vidal-Linan L, Bellas J, Etxebarria N, Nieto O, Beiras R.
Journal: Sci Total Environ (2014): 107
Temperature-driven switching of the catalytic activity of artificial glutathione peroxidase by the shape transition between the nanotubes and vesicle-like structures
Authors: Wang L, Zou H, Dong Z, Zhou L, Li J, Luo Q, Zhu J, Xu J, Liu J.
Journal: Langmuir (2014): 4013
Generation of superoxide anion radicals and platelet glutathione peroxidase activity in patients with schizophrenia
Authors: Dietrich-Muszalska A, Kwiatkowska A.
Journal: Neuropsychiatr Dis Treat (2014): 703
Clinical significance and therapeutic value of glutathione peroxidase 3 (GPx3) in hepatocellular carcinoma
Authors: Qi X, Ng KT, Lian QZ, Liu XB, Li CX, Geng W, Ling CC, Ma YY, Yeung WH, Tu WW, Fan ST, Lo CM, Man K.
Journal: Oncotarget. (2014)
Molecular cloning, characterization and expression profile of a glutathione peroxidase-like thioredoxin peroxidase (TPx) of the rodent malaria parasite Plasmodium berghei
Authors: Haselton KJ, David R, Fell K, Schulte E, Dybas M, Olsen KW, Kanzok SM.
Journal: Parasitol Int. (2014)
Model study using designed selenopeptides on the importance of the catalytic triad for the antioxidative functions of glutathione peroxidase
Authors: Takei T, Urabe Y, Asahina Y, Hojo H, Nomura T, Dedachi K, Arai K, Iwaoka M.
Journal: J Phys Chem B (2014): 492
The oxido-reductase enzyme glutathione peroxidase 4 (GPX4) governs Salmonella Typhimurium-induced neutrophil transepithelial migration
Authors: Agbor TA, Demma Z, Mrsny RJ, Castillo A, Boll EJ, McCormick BA.
Journal: Cell Microbiol (2014): 1339
Serum selenium concentration and glutathione peroxidase activity and selenium content in testes of Polish Konik horses from selenium--deficient area in North-Western Poland
Authors: Pilarczyk B, Tomza-Marciniak A, Stankiewicz T, Blaszczyk B, Gaczarzewicz D, Smugala M, Udala J, Tylkowska A, Kuba J, Ciesla A.
Journal: Pol J Vet Sci (2014): 165