Glutathione Reductase Fluorescent Kit

Produktnummer ABIN577651

Produktdetails

Target: Glutathione Reductase (GSR)

Nachweismethode: Fluorometric

Untere Nachweisgrenze: 9 µU/mL

Applikation: Biochemical Assay (BCA)

Verwendungszweck: The DetectX® Glutathione Reductase (GR) Fluorescent Activity kit is designed to quantitatively measure glutathione reductase (GR) activity in a variety of samples.

Marke: DetectX®

Proben: Serum, Plasma, Red Blood Cells, Cell Lysate

Spezifität: Sample Types validated: Serum, Plasma, RBCs and Cell Lysates

Sensitivität: 0.009 mU/mL

Produktmerkmale: The Glutathione Reductase (GR) Fluorescent Activity Kit determines GR activity by the amount of GSH generated from the reduction of GSSG with a non-fluorescent molecule, ThioStar®, that covalently binds the free thiol group on GSH to yield a highly fluorescent product. The most widely used procedure to measure GR is to monitor the oxidation of NADPH as a decrease in absorbance at 340nm. However many biological molecules absorb light at 340 nm, plus the detection system gives very low OD readings. The Arbor Assays' Glutathione Reductase Fluorescent Activity kit overcomes these problems of measuring GR activity by the direct detection of the GSH formed from oxidized glutathione. Glutathione reductase (GR) plays an indirect but essential role in the prevention of oxidative damage within the cell by helping to maintain appropriate levels of intracellular glutathione (GSH). GSH, in conjuction with the enzyme glutathione peroxidase (GP), is the acting reductant responsible for minimizing harmful hydrogen peroxide cellular levels. The regeneration of GSH is catalyzed by GR. GR is an ubiquitous 100-120 kDa dimeric flavoprotein that catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione, using ß-nicotinamide dinucleotide phosphate (NADPH) as the hydrogen donor. Molecules such as NADPH act as hydride donors in a variety of enzymatic processes. NADPH has been suggested to also act as an indirectly operating antioxidant, given its role in the re-reduction of GSSG to GSH and thus maintaining the antioxidative power of glutathione.

Bestandteile: Black Half Area 96 Well Plate
Glutathione Reductase Standard 40 μL Glutathione Reductase at 200 mU/mL in a special stabilizing solution.
ThioStar® Detection Reagent 1 vial ThioStar thiol detection substrate stored in a desiccator. Reconstitute with dry DMSO.
Dry DMSO 2 mL Dry Dimethyl sulfoxide solvent over molecular sieves. May be stored at room temperature.
Assay Buffer Concentrate 60 mL A 2x concentrated phosphate buffer containing detergents and stabilizers.
NADPH 1 vial Reduced ß-nicotinamide adenine dinucleotide 2'-phosphate freeze dried with stabilizers stored in a desiccator.
NADPH Diluent 5 mL A phosphate buffer containing detergents and stabilizers.
Oxidized Glutathione 3 mL Oxidized Glutathione (GSSG) in a special stabilizing solution.

Benötigtes Material: Repeater pipet with disposable tips capable of dispensing 15 μL and 25 μL.
Fluorescence 96 well plate reader capable of reading fluorescent emission at 510 nm, with excita- tion at 390 nm.
Software for converting raw relative fluorescent unit (FLU) readings from the plate reader and carrying out four parameter logistic curve (4PLC) fitting. Set plate parameters for a 96-well Corning Costar 3686 plate.
See: http://www.Ar- borAssays.com/resources/lit.asp for plate dimension data.
The Supplier has available for free download on our website an Excel spreadsheet useful in sub- tracting out sample thiol background at: http://www.arborassays.com/resources/lit.asp

Anwendungsinformationen

Applikationshinweise: This assay has been validated for human serum, EDTA and heparin plasma, and isolated erythro- cytes.
Most cell lysates should also be compatible.
Samples containing visible particulate should be centrifuged prior to using.
GR activity varies across tissues and species, however we expect this kit to measure GR activity from sources other than human.
The end user should evaluate re- coveries of GR activity in samples from other species being tested.

Testdauer: 1 h

Protokoll: A GR standard is provided to generate a standard curve for the assay and all samples should be read off the standard curve.
The kit utilizes a proprietary non-fluorescent molecule, ThioStar®, that will covalently bind to the free thiol group on GSH gen- erated in the reduction of oxidized glutathione (GSSG) to yield a highly fluorescent product.
After mixing the sample or standard with ThioStar® and incubating at room temperature, the fluorescent product is read at 510 nm in a fluorescent plate reader with excitation at 390 nm.
Background thiol content is read first after 5 minutes, followed by addition of GSSG and NADPH which uses the standard or sample GR to convert the oxidized glutathione, GSSG, into free GSH, which then reacts with the ThioStar® to yield the signal related to GR activity The activity of GR in the sample is calculated from the generated signal.
We have provided a 96 well plate for measure- ment but this assay is adaptable for higher density plate formats.
The end user should ensure that their HTS black plate is suitable for use with these reagents prior to running samples.

Aufbereitung der Reagenzien:

Allow the kit reagents to come to room temperature for 30 minutes.
We recommend that all stan- dards and samples be run in duplicate to allow the end user to accurately determine GR activity.
Ensure that all samples have reached room temperature and have been diluted as appropriate prior to running them in the kit.
Assay Buffer Preparation Prepare the Assay Buffer by diluting one part of the 2x Assay Buffer Concentrate 1:2 with one part deionized water.
It is stable for up to 3 months when stored at 4 °C.
Standard Preparation GR Standards are prepared by labeling six test tubes as #1 through #6.
Briefly spin vial of stan- dard in a microcentrifuge to ensure contents are at bottom of vial.
Pipet 390 μL of Assay Buffer into tube #1 and 200 μL into tubes #2 to #6.
Carefully add 10 μL of the Glutathione Reductase Standard to tube #1 and vortex completely.
Take 200 μL of the GR solution in tube #1 and add it to tube #2 and vortex completely.
Repeat these serial dilutions for tubes #3 through #6.
The concentration of GR in tubes 1 through 6 will be 5, 2.5, 1.25, 0.625, 0.3125, and 0.156 mU/mL.
Use all Standards within 2 hour of preparation.
Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Buffer Volume (μL) 390 200 200 200 200 200 Addition Stock Std 1 Std 2 Std 3 Std 4 Std 5 Volume of Addition (μL) 10 200 200 200 200 200 Final Conc (mU/ mL) 5 2.5 1.25 0.625 0.3125 0.156 ThioStar® Detection Reagent Remove the vial of ThioStar® Reagent from the desiccator and add 1.8 mL of the provided DMSO to the vial.
Vortex thoroughly.
Store any unused reconstituted Detection Reagent at 4 °C in the desiccator and use within 2 months.
NADPH Add 3 mL of the NADPH Diluent to the NADPH vial and vortex thoroughly.
Store any unused re- constituted NADPH at 4 °C for no more than 2 weeks.

Aufbereitung der Proben:

Any samples requiring larger dilutions or with GR activities outside the standard curve range should be diluted further with Assay Buffer to obtain readings within the standard curve. Serum and

Testdurchführung:

1. Use the plate layout sheet on the back page of the insert to aid in proper sample and standard identification. Set plate parameters for a 96-well Corning Costar 3686 plate.
   2. Pipet 25 μL of samples or standards into duplicate wells in the plate.
   3. Pipet 25 μL of Assay Buffer into duplicate wells as the Zero standard.
   4. Add 15 μL of the ThioStar® Detection Reagent to each well using a repeater pipet.
   5. Gently tap the sides of the plate to ensure adequate mixing of the reagents.
   6. Incubate at room temperature for 5 minutes.
   7. Read the fluorescent signal from each well in a plate reader capable of reading the fluorescent emission at 510 nm with excitation at 370-410 nm. Please contact your plate reader manufacturer for suitable filter sets. This data will be used to subtract any background thiol signal in samples.
   8. Add 25 μL of the Oxidized Glutathione to each of the wells using a repeater pipet.
   9. Add 25 μL of the NADPH to each of the wells using a repeater pipet. 10. Gently tap the sides of the plate to ensure adequate mixing of the reagents. 11. Incubate at room temperature for 15 minutes. 12. Read the fluorescent emission at 510 nm with excitation at 370-410 nm. Please contact your plate reader manufacturer for suitable filters.

Ergebnisberechnung:

The Supplier has available for free download on our website an Excel spreadsheet useful in sub- tracting out sample thiol background at: http://www.arborassays.com/resources/lit.asp After subtracting the background thiol FLU readings for each well from step 6, average the dupli- cate FLU readings for each standard and sample.
Create a standard curve by reducing the data using the 4PLC fitting routine on the plate reader, after subtracting the mean FLUs for the zero standard.
The activities obtained should be multiplied by the dilution factor to obtain neat sample values.
Or use the online tool from http://www.myassays.com/arbor-assays-glutathione-reductase-fluorescent-kit.assay to calculate the data. *

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Vorsichtsmaßnahmen: As with all such products, this kit should only be used by qualified personnel who have had labo- ratory safety instruction.
The complete insert should be read and understood before attempting to use the product.
Dimethyl sulfoxide is a powerful aprotic organic solvent that has been shown to enhance the rate of skin absorption of skin-permeable substances.
Wear protective gloves when using the solvent especially when it contains dissolved chemicals.
Reconstituted ThioStar® Detection Reagent should be stored at 4°C in the desiccator.
Allow to warm to room temperature prior to opening.
ThioStar will react with strong nucleophiles.
Buffers containing the preservatives sodium azide, Proclin™ and Kathon™ will react with the substrate.
Reconstituted ThioStar in DMSO stored at 4°C in the supplied desiccator can be used up to 2 months later.
The background on the reconstituted ThioStar will increase slowly over time but the increase will not affect the assay results obtained. activity standardization

Handhabung: The Glutathione Reductase standard used in this kit has been calibrated using an enzymatic method adapted from reference 4.

Lagerung: 4 °C

Informationen zur Lagerung: All components of this kit should be stored at 4°C until the expiration date of the kit. DMSO, when stored at 4°C, will freeze. Can be stored tightly capped at room temperature.

Referenzen

Lee, Kumar, Rani, Foster: "Role of antioxidant enzymes in redox regulation of N-methyl-D-aspartate receptor function and memory in middle-aged rats." in: Neurobiology of aging, Vol. 35, Issue 6, pp. 1459-68, (2014) (PubMed).

Stier, Bize, Habold, Bouillaud, Massemin, Criscuolo: "Mitochondrial uncoupling prevents cold-induced oxidative stress: a case study using UCP1 knockout mice." in: The Journal of experimental biology, Vol. 217, Issue Pt 4, pp. 624-30, (2014) (PubMed).

Alleva, Di Donato, Strafella, Staffolani, Nocchi, Borghi, Pignotti, Santarelli, Tomasetti: "Effect of ascorbic acid-rich diet on in vivo-induced oxidative stress." in: The British journal of nutrition, Vol. 107, Issue 11, pp. 1645-54, (2012) (PubMed).

Xie, Kang, Burris, Ferguson, Schauss, Nagarajan, Wu: "Açaí juice attenuates atherosclerosis in ApoE deficient mice through antioxidant and anti-inflammatory activities." in: Atherosclerosis, Vol. 216, Issue 2, pp. 327-33, (2011) (PubMed).

Antigendetails

Target: Glutathione Reductase (GSR)

Andere Bezeichnung: Glutathione Reductase (GSR Produkte)

Synonyme: GR Kit, ATGR2 Kit, EMB2360 Kit, glutathione reductase Kit, AI325518 Kit, D8Ertd238e Kit, Gr-1 Kit, Gr1 Kit, DDBDRAFT_0168952 Kit, DDBDRAFT_0231410 Kit, DDB_0168952 Kit, DDB_0231410 Kit, 2151 Kit, CG2151 Kit, DTR Kit, Dm-TrxR Kit, DmTR Kit, DmTrx Kit, DmTrxR Kit, DmTrxR-1 Kit, Dmel\\CG2151 Kit, Gr Kit, Trx Kit, TrxR Kit, TrxR-1 Kit, Trxr1 Kit, anon-WO03040301.185 Kit, anon-WO03040301.187 Kit, dTrxR Kit, dmtrxr-1 Kit, gr Kit, l(1)G0154 Kit, l(1)G0379 Kit, l(1)G0477 Kit, l(1)G0481 Kit, trxr-1 Kit, gor1 Kit, glutathione reductase, gro-2 Kit, glutathione reductase Kit, glutathione-disulfide reductase Kit, GR; GRase Kit, mitochondrial glutathione reductase Pgr1 Kit, Glutathione reductase (GR) (GRase) Kit, Thioredoxin reductase-1 Kit, glutathione-disulfide reductase family protein Kit, glutathione reductase 1 Kit, GR Kit, gor Kit, GSR Kit, GSR1 Kit, Gsr_2 Kit, Gsr Kit, GSHR1 Kit, gsr Kit, LbGR Kit, pgr1 Kit, GSHR2 Kit, GLR2 Kit, Bcen_2392 Kit, RPE_1989 Kit, HCAG_02219 Kit, SJAG_01162 Kit, Trxr-1 Kit, PF14_0192 Kit, POPTR_0001s14480g Kit, gsr1 Kit

Hintergrund: Glutathione reductase (GR) plays an indirect but essential role in the prevention of oxidative dam- age within the cell by helping to maintain appropriate levels of intracellular glutathione (GSH). GSH, in conjuction with the enzyme glutathione peroxidase (GP), is the acting reductant respon- sible for minimizing harmful hydrogen peroxide cellular levels1. The regeneration of GSH is cata- lyzed by GR2. GR is an ubiquitous 100-120 kDa dimeric flavoprotein that catalyzes the reduction of oxidized glutathione (GSSG) to reduced glutathione, using ß-nicotinamide dinucleotide phos- phate (NADPH) as the hydrogen donor3. Molecules such as NADPH act as hydride donors in a variety of enzymatic processes. NADPH has been suggested to also act as an indirectly operating antioxidant, given its role in the re-reduction of GSSG to GSH and thus maintaining the antioxida- tive power of glutathione. The general GR reaction is shown below: Glutathione Reductase GSSG 2 GSH NADPH + H+ NADP+ The most widely used procedure to measure GR is to monitor the oxidation of NADPH as a de- crease in absorbance at 340nm4. However this method suffers from the absorbance of many biological molecules at 340nm. This DetectX® assay determines GR activity by directly measuring the amount of GSH generated from the reduction of GSSG by reacting the GSH with a non-fluorescent molecule, ThioStar®, to covalently bind the free thiol group on GSH and yield a highly fluorescent product