359963 | Glutathione Reductase Assay Kit II

359963
  
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      A sensitive spectrophotometric assay (340 nm) for the measurement of glutathione reductase (GR) activity in plasma, erythrocyte lysates, tissue homogenates, and cell lysates. Assay range: 20-255 nmol/min/ml. Contains sufficient reagents for 96 tests.
      Référence 359963
      Famille de marques Calbiochem®
      Références bibliographiques
      Références bibliographiques Foyer, C.H., et al. 1994. Physiol. Plant. 92, 696.
      Baillie, T.A. and Slatter, J.G. 1991. Acc. Chem. Res. 24, 264.
      Inoue, M., et al. 1987. Journal of Protein Chemistry 6, 207.
      Carlberg, I., et al. 1985. Methods Enzymol. 113, 484.
      Informations produit
      Méthode de détection Colorimetric
      Forme 96 Tests
      Format 96-well plate
      Contenu du kit Assay Buffer, Sample Buffer, Glutathione Reductase (control), GSSG, NADPH, 96 Well Plate, Plate Cover
      Témoin positif Glutathione reductase (Baker's Yeast)
      Applications
      Informations biologiques
      Gamme d'essais 20-255 nmol/min/ml
      Temps de l'essai 2 h
      Type d'échantillon Plasma, erythrocyte lysates, tissue homogenates, and cell lysates
      Informations physico-chimiques
      Dimensions
      Informations sur les matériaux
      Informations toxicologiques
      Informations de sécurité selon le SGH
      Informations sur la sécurité
      Notifications sur l'utilisation du produit
      Informations relatives au stockage et à l'expédition
      Code Expé Blue Ice Only
      Toxicité Standard Handling
      Stockage -20°C
      Conditions de stockage Upon arrival store the entire contents of the kit at -20°C.
      Ne pas congeler No
      Informations sur l'emballage
      Informations sur le transport
      Information complémentaire
      Contenu du kit Assay Buffer, Sample Buffer, Glutathione Reductase (control), GSSG, NADPH, 96 Well Plate, Plate Cover
      Caractéristiques

      Documentation

      FDS

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      TitreNuméro de lot
      359963

      Références bibliographiques

      Aperçu de la référence bibliographique
      Foyer, C.H., et al. 1994. Physiol. Plant. 92, 696.
      Baillie, T.A. and Slatter, J.G. 1991. Acc. Chem. Res. 24, 264.
      Inoue, M., et al. 1987. Journal of Protein Chemistry 6, 207.
      Carlberg, I., et al. 1985. Methods Enzymol. 113, 484.
      Protocole Utilisateur

      Révision 05-September-2008 RFH
      Forme 96 Tests
      Format 96-well plate
      Méthode de détection Colorimetric
      Stockage Upon arrival store the entire contents of the kit at -20°C.
      Contexte Glutathione (GSH) is a tripeptide widely distributed in both plants and animals. GSH serves as a nucleophilic co-substrate to GSH transferases in the detoxification of xenobiotics and is an essential electron donor to GSH peroxidases in the reduction of hydroperoxides. GSH is also involved in amino acid transport across membranes. Glutathione reductase (GR, EC 1.6.4.2) is a flavoprotein that catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG) to GSH. This enzyme is essential for the GSH redox cycle that maintains adequate levels of reduced cellular GSH. A high GSH/GSSG ratio is essential for protection against oxidative stress.
      Principes de l'essai The Calbiochem® Glutathione Reductase Assay Kit measures glutathione reductase activity by measuring the rate of NADPH oxidation:

      Figure 1: Equation 1


      The oxidation of NADPH to NADP+ is accompanied by a decrease in absorbance at 340 nm and is directly proportional to the glutathione reductase activity in the sample. The assay can be used to measure glutathione reductase activity in plasma, erythrocyte lysates, tissue homogenates, and cell lysates.
      Matériel fourni • 10X Assay Buffer (Kit Component No. KP31677-3ML): 1 vial
      • 10X Sample Buffer (Kit Component No. KP31678-3ML): 1 vial
      • Glutathione Reductase (Control) (Kit Component No. KP31679-50UL): 1 vial
      • GSSG (Kit Component No. KP31680-2.5ML): 1 vial
      • NADPH (Kit Component No. KP31681-1EA): 3 vials
      • 96-Well Plate (Kit Component No. KP31625-1EA): 1 plate
      • Plate Sealer (Kit Component No. KP31626-1EA): 1 plate sealer
      Matériel requis, mais non fourni A plate reader with a 340 nm filter
      An adjustable pipettor and a repeat pipettor
      A source of pure water. Glass distilled water or HPLC-grade water is acceptable
      PBS
      Homogenization buffer (tissue and cell preparation)
      Précautions et recommandations Please read these instructions carefully before beginning this assay.
      It is recommended to take appropriate precautions when using the kit reagents (i.e., lab coat, gloves, eye goggles, etc.) as some of them can be harmful.
      Pipetting Hints
      a. It is recommended that a repeat pipettor be used to deliver reagents to the wells. This saves time and helps to maintain more precise incubation times
      b. Use different tips to pipet GSSG, enzyme, and NADPH
      c. Before pipetting each reagent, equilibrate the pipet tip (i.e., fill the tip and gently expel the contents several times, repeat several times).
      d. Do not expose the pipet tip to the reagent(s) already in the well.
      Préparation 1. Tissue Homogenates Prior to dissection, either perfuse tissue or rinse tissue with a phosphate buffered saline (PBS) solution, pH 7.4, to remove any red blood cells and clots. Homogenize the tissue on ice in 5-10 ml cold homogenization buffer (i.e., 50 mM potassium phosphate, pH 7.0, containing 1 mM EDTA) per gram tissue. Centrifuge at 10,000 x g for 15 min at 4°C. 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. 2. Cell Lysate Collect cells by centrifugation (i.e., 1,000-2,000 x g for 10 min at 4°C). For adherent cells, do not harvest using proteolytic enzymes; rather use a rubber policeman. Homogenize cell pellet in cold homogenization buffer (i.e., 50 mM potassium phosphate, pH 7.0, containing 1 mM EDTA). Centrifuge at 10,000 x g for 15 min at 4°C. 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. 3. Plasma and Erythrocyte lysate Collect blood using an anticoagulant such as heparin, citrate, or EDTA. 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. Remove the white buffy layer (leukocytes) and discard. Lyse the erythrocytes (red blood cells) in 4 times its volume with ice-cold HPLC-grade water. Centrifuge at 10,000 x g for 15 min at 4°C. 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.
      Préparation des réactifs • Assay Buffer: Dilute 2 ml 10X Assay Buffer concentrate with 18 ml HPLC-grade water. This final diluted Assay Buffer (100 mM potassium phosphate, pH 7.0) should be used in the assay. When stored at 4°C, this diluted Assay Buffer is stable for at least 2 months. • Sample Buffer: Dilute 3 ml of 10X sample buffer concentrate with 21 ml of HPLC-grade water. This final diluted Sample Buffer (25 mM potassium phosphate, pH 7.5, containing 1 mM EDTA and 0.1% BSA) should be used to dilute the Glutathione Reductase (Control) and glutathione reductase samples prior to assaying. When stored at 4°C, this diluted Sample Buffer is stable for up to 2 months. • Glutathione Reductase (Control): This vial contains a solution of glutathione reductase (from Baker's Yeast). To avoid repeated freezing and thawing, the glutathione reductase (Control) should be aliquoted into several small vials and stored at -20°C. Prior to use, transfer 10 µl of the supplied enzyme to another vial and dilute with 990 µl diluted Sample Buffer and keep on ice. The diluted enzyme is stable for up to 4 h on ice. A 20 µl aliquot of this diluted enzyme per well causes a decrease of ~0.04 absorbance units/min under the standard assay conditions described in the Detailed Protocol. • GSSG: This vial contains a 9.5 mM solution of GSSG and should be stored at -20°C when not being used. The reagent is ready to use as supplied. NADPH: The vials contain a lyophilized powder of NADPH. 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 up to 2 days. [NOTE: Do not freeze the reconstituted reagent.]
      Protocole détaillé 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.

      1. Background or Non-enzymatic Wells: Add 120 µl diluted Assay Buffer and 20 µl GSSG to 3 wells.
      2. Positive Control Wells (Baker's yeast glutathione reductase): Add 100 µl diluted Assay Buffer, 20 µl GSSG, and 20 µl diluted Glutathione Reductase (Control) to 3 wells.
      3. Sample Wells: Add 100 µl diluted Assay Buffer, 20 µl GSSG, and 20 µl sample to 3 wells. To obtain reproducible results, the amount of glutathione reductase added to the well should cause an absorbance decrease between 0.008 and 0.1/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 50 µl NADPH to all the wells. Make sure to note the precise time you started and add the NADPH as quickly as possible.
      5. Carefully shake the 96-well plate for a few seconds 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.
      Données d'exemple 1. Determine the change in absorbance (ΔA₃₄₀) per min by 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 Baker's yeast glutathione reductase) -or- selecting two points on the linear portion of the curve and determining the change in absorbance during that time using the following equation:

      Figure 2: Equation 2

      2. Determine the rate of ΔA₃₄₀/min for the background or non-enzymatic wells and subtract this rate from that of the sample wells. 3. Use the following formula to calculate the glutathione reductase 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 min 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 pathlength of the solution in the well (0.6 cm).

      Figure 4: Activity of Baker's Yeast Glutathione Reductase

      Remarques sur la sensibilité 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 reductase activity between 20-255 nmol/min/ml can be assayed without further dilution or concentration. This glutathione reductase activity is equivalent to an absorbance decrease of 0.008 to 0.1 per min.
      Gamme d'essais 20-255 nmol/min/ml
      Marques déposées Calbiochem® is a registered trademark of EMD Chemicals, Inc.
      Interactive Pathways™ is a trademark of EMD Chemicals, Inc.

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