353919 | Glutathione Peroxidase Assay Kit

353919
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      Overview

      Key Spec Table

      Detection Methods
      Colorimetric

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      353919-1KIT
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      Discontinued
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          Description
          Overview A sensitive spectrophotometric assay (340 nm) for the measurement of glutathione peroxidase (GPx) activity in plasma, erythrocyte lysates, tissue homogenates, and cell lysates. Assay range: 50-344 nmol/min/ml. Contains sufficient reagents for 96 tests.
          Catalogue Number 353919
          Brand Family Calbiochem®
          References
          References Ursini, F., et al. 1985. Biochim. Biophys. Acta 839, 62.
          Forstrom, J.W., et al. 1978. Biochem. 17, 2639.
          Paglia, D.E., et al. 1967. J. Lab. Clin. Med. 70, 158.
          Product Information
          Detection method Colorimetric
          Form 96 Tests
          Format 96-well plate
          Kit contains Assay Buffer, Sample Buffer, Glutathione Peroxidase (Control), Co-Substrate Mixture, Cumene Hydroperoxide, 96 Well Plate, Plate Cover, and a user protocol.
          Positive control Bovine erythrocyte glutathione peroxidase
          Applications
          Biological Information
          Assay range 50-344 nmol/min/ml
          Assay time 2 h
          Sample Type Cells, tissues, plasma
          Physicochemical Information
          Dimensions
          Materials Information
          Toxicological Information
          Safety Information according to GHS
          Safety Information
          Product Usage Statements
          Storage and Shipping Information
          Ship Code Blue Ice Only
          Toxicity Standard Handling
          Storage -20°C
          Storage Conditions Upon arrival store the entire contents of the kit at -20°C.
          Do not freeze No
          Packaging Information
          Transport Information
          Supplemental Information
          Kit contains Assay Buffer, Sample Buffer, Glutathione Peroxidase (Control), Co-Substrate Mixture, Cumene Hydroperoxide, 96 Well Plate, Plate Cover, and a user protocol.
          Specifications

          Documentation

          MSDS

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          Certificates of Analysis

          TitleLot Number
          353919
          User Protocol

          Revision 10-April-2014 JSW
          Form 96 Tests
          Format 96-well plate
          Detection method Colorimetric
          Storage Upon arrival store the entire contents of the kit at -20°C.
          Background Glutathione peroxidase (glutathione peroxidase) catalyzes the reduction of hydroperoxides, including hydrogen peroxide, by reduced glutathione and functions to protect the cell from oxidative damage. With the exception of phospholipid-hydroperoxide Glutathione peroxidase, a monomer, all of the glutathione peroxidase enzymes are tetramers of four identical subunits. Each subunit contains a selenocysteine in the active site that participates directly in the two-electron reduction of the peroxide substrate. The enzyme uses glutathione as the ultimate electron donor to regenerate the reduced form of the selenocysteine.
          Principles of the assay The Calbiochem® Glutathione Peroxidase Assay Kit measures glutathione peroxidase (GPx) activity indirectly by a coupled reaction with glutathione reductase (GR). Oxidized glutathione (GSSG), produced upon reduction of hydroperoxide by glutathione peroxidase, is recycled to its reduced state by glutathione reductase and NADPH:

          Figure 1: Equation 1


          The oxidation of NADPH to NADP+ is accompanied by a decrease in absorbance at 340 nm. Under conditions in which the glutathione peroxidase activity is rate limiting, the rate of decrease in the A₃₄₀ is directly proportional to the glutathione peroxidase activity in the sample. The assay can be used to measure all of the glutathione-dependent peroxidases in plasma, erythrocyte lysates, tissue homogenates, and cell lysates.
          Materials provided • 10X Assay Buffer (Kit Component No. KP31672-3ML): 1 vial, 3 ml
          • 10X Sample Buffer (Kit Component No. KP31673-3ML): 1 vial, 3 ml
          • Glutathione Peroxidase (Control) (Kit Component No. KP31674-50UL): 1 vial, 50 µl
          • Co-Substrate Mixture (Kit Component No. KP31675-1EA): 2 vials
          • Cumene Hydroperoxide (Kit Component No. KP31676-2.5ML): 1 vial, 2.5 ml
          • 96-Well Plate and Plate Sealer (Kit Component No. KP31625-1EA): 1 plate, 1 plate sealer
          Materials Required but not provided A plate reader with a 340 nm filter
          Adjustable pipettors and a repeat pipettor
          A source of pure water. Glass distilled water or HPLC-grade water is acceptable
          PBS
          Homogenization buffer (cell and tissue preparation)
          Precautions and recommendations Please read these instructions carefully before beginning this assay.
          Pipetting Hints
          a. It is recommended that a repeating pipettor be used to deliver dilute Assay Buffer, co-substrate mixture, and cumene hydroperoxide to the wells. This saves time and helps to maintain more precise incubation times
          b. Use different tips to pipet the dilute Assay Buffer, co-substrate mixture, enzymes, and cumene hydroperoxide.
          c. Before pipetting each reagent, equilibrate the pipet tip in that reagent (i.e., slowly fill the tip and gently expel the contents, repeat several times)
          d. Do not expose the pipet tip to the reagent(s) already in the well.
          Preparation • Tissue Homogenate: 1. Prior to dissection, perfuse tissue with a PBS (phosphate buffered saline) solution, pH 7.4, containing 0.16 mg/ml heparin to remove any red blood cells and clots. 2. Homogenize the tissue in 5-10 ml cold homogenization buffer (i.e., 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, and 1 mM DTT) per gram tissue. 3. Centrifuge at 10,000 x g for 15 min at 4°C. 4. Remove the supernatant for assay and store on ice. If not assaying on the same day, freeze the sample at -80°C. The sample will be stable for at least one month. • Cell Lysate: 1. Collect cells by centrifugation (i.e., 1000-2000 x g for 10 min at 4°C). For adherent cells, do not harvest using proteolytic enzymes; rather use a rubber policeman. 2. Homogenize cell pellet in cold homogenization buffer (i.e., 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, and 1 mM DTT). 3. Centrifuge at 10,000 x g for 15 min at 4°C. 4. Remove the supernatant for assay and store on ice. If not assaying on the same day, freeze the sample at -80°C. The sample will be stable for at least one month. • Plasma and Erythrocyte Lysate: 1. Collect blood using an anticoagulant such as heparin, citrate, or EDTA. 2. Centrifuge the blood at 700-1,000 x g for 10 min at 4°C. Pipet off the top yellow plasma layer without disturbing the white buffy layer. Store plasma on ice until assaying or freeze at -80°C. The plasma sample will be stable for at least one month. 3. Remove the white buffy layer (leukocytes) and discard. 4. Lyse the erythrocytes (red blood cells) in 4 volumes ice-cold HPLC-grade water. 5. Centrifuge at 10,000 x g for 15 min at 4°C. 6. Collect the supernatant (erythrocyte lysate) for assaying and store on ice. If not assaying the same day, freeze at -80°C. The sample will be stable for at least one month. Note: It has been reported that heme peroxidase activity of hemoglobin can lead to falsely elevated Glutathione peroxidase activity in erythrocyte lysates. There was no significant effect in the Glutathione peroxidase activity when assayed with cumene hydroperoxide as the substrate. Therefore, it is not necessary to treat the sample with Drabkin's Reagent (potassium ferricyanide/potassium cyanide) to convert hemoglobin to cyanmethemoglobin before assaying.
          Reagent preparation • 1X Assay Buffer: Dilute 2 ml Assay Buffer concentrate with 18 ml HPLC-grade water. This final diluted Assay Buffer (50 mM Tris-HCl, pH 7.6, containing 5 mM EDTA) should be used in the assay. When stored at 4°C, this diluted Assay Buffer is stable for at least two months. • 1X Sample Buffer: Dilute 2 ml Sample Buffer concentrate with 18 ml HPLC-grade water. This final dilute Sample Buffer (50 mM Tris-HCl, pH 7.6, containing 5 mM EDTA and 1 mg/ml BSA) should be used to dilute the Glutathione Peroxidase (Control) and the glutathione peroxidase samples prior to assaying. When stored at 4°C, this diluted Sample Buffer is stable for at least one month. • Glutathione Peroxidase (Control): This vial contains a solution of bovine erythrocyte glutathione peroxidase. To avoid repeated freezing and thawing, the Glutathione Peroxidase (Control) should be dispensed into small aliquots and stored at -20°C. Prior to use, transfer 10 µl of the Glutathione Peroxidase (Control) to another vial and dilute with 490 µl diluted Sample Buffer and keep on ice. The diluted Glutathione Peroxidase (Control) is stable for 4 h on ice. A 20 µl aliquot of this diluted Glutathione Peroxidase (Control) per well causes a decrease of approximately 0.051 absorbance unit/min under the standard assay conditions described in the Detailed Protocol. • Co-Substrate Mixture: These vials contain a lyophilized powder of NADPH, glutathione, and glutathione reductase. Each reconstituted vial will be enough reagent for 40 wells. Reconstitute the number of vials that you will need by adding 2 ml HPLC-grade water to each vial and vortex well. The reconstituted reagent should be kept at 25°C while assaying and then stored at 4°C. If stored at 4°C, the reconstituted reagent is stable for 2 days. Do not freeze the reconstituted reagent. • Cumene Hydroperoxide: This vial contains a solution of cumene hydroperoxide and should be stored at -20°C when not being used. The reagent is ready to use as supplied.
          Detailed protocol Notes:
          The final volume of the assay is 190 µl in all the wells.
          It is not necessary to use all the wells on the plate at one time.
          The assay temperature is 25°C.
          Use the diluted Assay Buffer in the assay.
          Monitor the decrease in absorbance at 340 nm using a plate reader.
          There is no specific pattern for using the wells on the plate. However, it is necessary to have 3 wells designated as non-enzymatic or background wells. The absorbance rate of these wells must be subtracted from the absorbance rate measured in the glutathione peroxidase sample and control wells. We suggest that there be at least 3 wells designated as positive controls.

          1. Background or Non-enzymatic Wells: Add 120 µl diluted Assay Buffer and 50 µl Co-Substrate Mixture to 3 wells.
          2. Positive Control Wells (bovine erythrocyte glutathione peroxidase): Add 100 µl diluted Assay Buffer, 50 µl of Co-Substrate Mixture, and 20 µl diluted Glutathione Peroxidase (Control) to 3 wells.
          3. Sample Wells: Add 100 µl diluted Assay Buffer, 50 µl of Co-Substrate Mixture, and 20 µl sample to 3 wells. To obtain reproducible results, the amount of glutathione peroxidase added to the well should cause an absorbance decrease between 0.02 and 0.135/min. When necessary, samples should be diluted with diluted Sample Buffer or concentrated with an Amicon centrifuge concentrator with a molecular weight cut-off of 10,000 to bring the enzymatic activity to this level.

          Note: The amount of sample added to the well should always be 20 µl. To determine if an additional sample control should be performed see the Interferences section.

          4. Initiate the reactions by adding 20 µl Cumene Hydroperoxide to all the wells being used. Make sure to note the precise time the reaction is initiated and add the Cumene Hydroperoxide as quickly as possible.
          5. Carefully shake the plate for a few s to mix.
          6. Read the absorbance once every minute at 340 nm using a plate reader to obtain at least 5 time points.
          Note: The initial absorbance of the sample wells should not be above 1.2 or below 0.5.
          7. Calculating the results: determination of the reaction rate
          a. Determine the change in absorbance (ΔA340) per min by:
          i. Plotting the absorbance values as a function of time to obtain the slope (rate) of the linear portion of the curve (a graph is shown below using bovine erythrocyte glutathione peroxidase) -or-
          ii. Select two points on the linear portion of the curve and determine the change in absorbance during that time using the following equation:

          Figure 2: Equation 2


          b. Determine the rate of ΔA340/min. for the background or non-enzymatic wells and subtract this rate from that of the sample wells.
          c. Use the following formula to calculate the Glutathione peroxidase activity. The reaction rate at 340 nm can be determined using the NADPH extinction coefficient of 0.00373 µM-1*. One unit is defined as the amount of enzyme that will cause the oxidation of 1.0 nmol of NADPH to NADP+ per minute at 25°C.

          Figure 3: Equation 3


          * The actual extinction coefficient for NADPH at 340 nm is 0.00622 µM-1cm-1. This value has been adjusted for the path length of the solution in the well (0.6 cm).
          Assay characteristics and examples

          Figure 4: Activity of Bovine Erythrocyte Glutathione Peroxidase

          Assay Range 50-344 nmol/min/ml
          Assay Range Notes The dynamic range of the assay is limited only by the accuracy of the absorbance measurement. Most plate readers are linear to an absorbance of 1.2. Samples containing glutathione peroxidase activity between 50-344 nmol/min/ml can be assayed without further dilution or concentration. This glutathione peroxidase activity is equivalent to an absorbance decrease of 0.02 to 0.135 per min.
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