OxiSelect™ Glutathione Reductase Assay Kit

Produktnummer ABIN2345145

Kurzübersicht für OxiSelect™ Glutathione Reductase Assay Kit (ABIN2345145)

Reaktivität: Others

Applikation: Biochemical Assay (BCA)

Proben: Cell Lysate, Tissue Samples, Plasma

Analytische Methode: Quantitative

Produktdetails

Verwendungszweck: The OxiSelect™ Glutathione Reductase Assay Kit is a quantitative assay for measuring the glutathione reductase activity within a sample.

Marke: OxiSelect™

Sensitivität: 0.6 mU/mL

Produktmerkmale: OxiSelect™ Glutathione Reductase Assay Kit is a quantitative assay for measuring glutathione reductase activity within plasma, erythrocytes, tissues, and cell lysates. Glutathione reductase activity is defined as 1 unit of enzyme reducing 1 μmole oxidized glutathione (GSSG) per minute at pH 7.6 and 25 °C. The kit employs a simple enzymatic recycling reaction for glutathione quantification where the reduction of a chromagen is correlated to glutathione reductase enzymatic activity. The kit has a detection sensitivity limit of approximately 0.6 mU/mL. Each kit provides sufficient reagents to perform up to 100 assays, including standard curve and unknown samples.

Bestandteile:

1. Glutathione Reductase Standard : One 20 μL amber tube.
2. Glutathione Disulfide (GSSG) : One 5 mL bottle of a 10 mM solution.
3. Chromogen : One 0.5 mL amber tube.
4. Assay Buffer (5X) : Two 25 mL bottles.

Box 2 (shipped on blue ice packs)

Benötigtes Material:

1. 96-well microtiter plate
2. Distilled or deionized water
3. 1X PBS
4. 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips
5. 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
6. Conical tubes and bottles for sample and buffer preparation
7. Centrifuge and/or microfuge
8. Sonicator or tissue homogenizer
9. Multichannel micropipette reservoirs
10. Ethanol
11. Microplate reader capable of reading 405 nm 3

Anwendungsinformationen

Applikationshinweise: Optimal working dilution should be determined by the investigator.

Kommentare:

• Detection sensitivity limit of approximately 0.6 mU/mL
• Quantitative assay for measuring glutathione reductase activity within plasma, erythrocytes, tissues, and cell lysates
• Sufficient reagents to perform up to 100 assays, including standard curve and unknown samples

Protokoll: Glutathione Reductase reduces oxidized glutathione (GSSG) to reduced glutathione (GSH) in the presence of NADPH. Subsequently, the chromogen reacts with the thiol group of GSH to produce a colored compound that absorbs at 405 nm . The glutathione reductase content in unknown samples is determined by comparison with the predetermined glutathione reductase standard curve. The rate of chromophore production is proportional to the concentration of glutathione reductase activity within the sample. The rate can be determined from the absorbance change over time.

Aufbereitung der Reagenzien:

1X Assay Buffer: Prepare 1X Assay Buffer by adding 200 mL of deionized water to 50 mL of the 5X Assay Buffer. Mix thoroughly until homogeneous. Use this buffer for preparing kit reagents. Store at 4 °C when not in use. Chromogen: Prepare the Chromogen just before use and prepare only enough for immediate applications. Dilute the Chromogen stock 1:15 with 1X Assay Buffer (eg. Add 200 μL of Chromogen stock to 2.8 mL of 1X Assay Buffer. Vortex thoroughly. 1X NADPH: Prepare 1X NADPH by diluting the stock solution 1:50 with 1X Assay Buffer. Vortex the stock tube thoroughly prior to preparing. Prepare only enough for immediate applications (eg. Add 25 μL of NADPH stock to 1.225 mL Assay Buffer).

Aufbereitung der Proben:

These preparation protocols are intended as a guide for preparing known samples. The user may need to adjust the sample treatment accordingly. All samples should be assayed immediately or stored for up to 1-2 months at -80 °C. A trial assay with a representative test sample should be assayed to determine the samples compatibility with the dynamic range of the standard. The assay can be used on cell culture supernatants, plasma, erythrocytes, tissues, and cell lysates. High levels of interfering substances may cause variations in results. Run proper controls as necessary. Always run a standard curve with samples. Notes: Thiol compounds, such as cysteine, dithiothreitol (DTT), or β-mercaptoethanol can interfere with the assay by competing with GSH for binding to the chromogen. In addition, N- ethylmaleimide or other thiol alkylating reagents should also be avoided because they will interfere with glutathione reductase and GSH. Make serial dilutions of samples as necessary to obtain a quantifiable change in absorbance readings over time. Samples containing reduced glutathione (GSH) should be accounted for by running the sample without Glutathione Disulfide (GSSG) and subtracting these background values from a treated sample. A kinetic assay is recommended because it is more precise than an end point assay. Plasma: Add blood sample to a blood collection tube with an anticoagulant such as heparin, EDTA, or sodium citrate. Centrifuge at 3,000 rpm for 10-15 minutes at 4 °C. Remove the upper yellow plasma supernatant layer without disturbing the white buffy coat (leukocytes). Store on ice if assaying immediately or freeze at -80 °C for up to 1-2 months. Dilute the plasma with 1X Assay Buffer before testing. Erythrocytes: Add blood sample to a blood collection tube with an anticoagulant such as heparin, EDTA, or sodium citrate. Centrifuge the blood at 3,000 rpm for 10-15 minutes at 4 °C. Remove the white interface buffy coat (leukocytes) and yellow plasma layers and discard. Wash the erythrocytes in cold PBS or saline. Determine the packed cell volume and add 4 cell volumes of 4 cold deionized water to lyse the cells. Centrifuge at 10,000-12,000 rpm for 10 minutes at 4 °C. Collect the supernatant. Store on ice if assaying immediately or freeze at -80 °C for up to 1-2 months. Dilute with 1X Assay Buffer before testing. 6Cell Lysates: Wash adherent cells at 2-6 x 10 cells with 1X PBS. Harvest adherent cells with a rubber policeman, or gentle trypsinization. Centrifuge adherent or suspension cells at 500-1000 rpm for 5 minutes at 4 °C. Remove supernatant and wash cells in cold PBS. Repeat centrifugation and remove solution. Immediately resuspend the cell pellet with 200-500 μL ice-cold 1X Assay 6 Buffer for a cell number of 1-5 x 10 cells. Mix thoroughly. Homogenize or sonicate cell suspension and store on ice until use. Transfer the suspension to a microfuge tube and centrifuge at 12,000 rpm for 5 minutes at 4 °C. Collect the supernatant. Store on ice if used immediately or freeze at -80 °C for up to 1-2 months. Dilute with 1X Assay Buffer as necessary before testing. Tissue Lysate: Perfuse or wash the tissue to remove blood cells and clots with a cold PBS/1 mM EDTA. Homogenize the tissue thoroughly with cold isotonic saline solution of 1X PBS with 0.16 mg/mL heparin to prevent coagulation. Blot the tissue dry and weigh. Add ice-cold PBS/1 mM EDTA (~1 mL/100 mg tissue) and homogenize using a glass pestle. Centrifuge the homogenate at 12,000 rpm for 15 minutes at 4 °C. Collect the supernatant. Store on ice if used immediately or freeze at -80 °C for up to 1-2 months. Dilute with 1X Assay Buffer as necessary before testing.

Testdurchführung:

1. Prepare and mix all reagents thoroughly before use. Prepare the glutathione reductase standards simultaneously with the samples so they may be assayed together. Each sample, including unknown and standard, should be assayed in duplicate or triplicate.
2. In a 96-well plate, add 25 μL of the 1X NADPH solution to each well to be tested.
3. Add 100 μL of the prepared glutathione reductase standards or samples to each well to be tested.
4. Add 50 μL of the 1X Chromogen and mix briefly.
5. Ensure that the plate reader is prepared for a kinetic assay and is set to read at 405 nm.
6. Add 25 μL of the Glutathione Disulfide (GSSG) solution and mix briefly. Immediately begin recording the absorbance at 405 nm at 1 minute intervals for 10 minutes. The reaction may be run longer if necessary. If using all the wells within the plate at one time, then it may be necessary to record the absorbance at 2 minute intervals.
7. Calculate the concentration of standards and samples. See Calculation of Results below. Calculation of Results
   1. First, determine the average of the replicate absorbance readings for each glutathione reductase standard, sample, and negative control for every time point taken.
   2. Graph the average of each standard, sample, and background absorbance at 405nm against incubation time. Determine the slope for each value from the linear portion of each curve. See Figure 2 under Example of Results below.
   3. Next, subtract the background slope from the slope of the standards and samples.
   4. Plot the net slopes of the glutathione reductase standards against the microunits/mL concentration of glutathione reductase. See Figure 3 under Example of Results below.
   5. Compare the net slopes of the samples with the standard curve from Figure 3 and determine the microunits/mL concentration of glutathione reductase for each sample. Note: Remember that the standard and samples are diluted 1:2 dilution within the final assay reaction protocol.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Handhabung: Avoid multiple freeze/thaw cycles.

Lagerung: 4 °C/-80 °C

Informationen zur Lagerung: Upon receipt, store the NADPH at -80°C. Prepare single use aliquots and avoid multiple freeze/thaw cycles. Store the remaining kit components at 4°C.

Referenzen

Kim, Lee, Cheon, Nam, Oh, Kim: "Antioxidant and hepatoprotective effects of fermented red ginseng against high fat diet-induced hyperlipidemia in rats." in: Laboratory animal research, Vol. 32, Issue 4, pp. 217-223, (2017) (PubMed).

Arrabal, Lucena, Canduela, Ramos-Uriarte, Rivera, Serrano, Pavón, Decara, Vargas, Baixeras, Martín-Rufián, Márquez, Fernández-Llébrez, De Roos, Grandes, Rodríguez de Fonseca, Suárez: "Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle." in: PLoS ONE, Vol. 10, Issue 12, pp. e0145244, (2015) (PubMed).

Antigendetails

Hintergrund: Glutathione reductase is a homodimeric enzyme that is a member of the flavoprotein disulfide oxidoreductases. It has an indirect impact in the prevention of oxidative damage in cells by helping to maintain intracellular reduced glutathione (GSH). Thus, measuring the activity of the enzyme is an indicator of oxidative stress. It is a ubiquitous enzyme that catalyzes the NADPH dependent reduction reaction of oxidized glutathione (GSSG) to reduced glutathione (GSH). Oxidized glutathione is reduced through a multi-step reaction in which glutathione reductase is reduced by NADPH, which in turn reacts with a GSSG molecule. This creates a disulfide interchange reaction that creates two GSH molecules and restores glutathione reductase to its oxidized form. Regenerated GSH is available to detoxify hydrogen peroxide. Maintenance of GSH is vital in oxidation-reduction processes as well as detoxification of hydrogen peroxide and organic peroxides brought on by inflammation in cells.