Glutathione Fluorescent Detection Kit

• Reproducibility:

  Sample GSH Conc. (uM) %CV Free Total Free Total 1 1.27 2.30 4.0 4.7 2 2.00 3.80 3.1 4.7 3 8.33 9.77 4.6 2.7 4 3.89 4.45 3.0 2.3 Sample GSH Conc. (uM) %CV Sample GSH Conc. (uM) %CV Free Total Free Total Free Total Free Total 1 1.27 2.30 4.0 4.7 1 1.27 2.30 4.0 4.7 2 2.00 3.80 3.1 4.7 2 2.00 3.80 3.1 4.7 3 8.33 9.77 4.6 2.7 3 8.33 9.77 4.6 2.7 4 3.89 4.45 3.0 2.3 4 3.89 4.45 3.0 2.3 Sample GSH Conc. (uM) %CV Free Total Free Total 1 1.30 2.40 8.6 8.3 2 1.83 3.57 14.7 10.0 3 9.38 11.67 6.0 6.0 4 4.89 5.89 7.2 8.0 Sample GSH Conc. (uM) %CV Sample GSH Conc. (uM) %CV Free Total Free Total Free Total Free Total 1 1.30 2.40 8.6 8.3 1 1.30 2.40 8.6 8.3 2 1.83 3.57 14.7 10.0 2 1.83 3.57 14.7 10.0 3 9.38 11.67 6.0 6.0 3 9.38 11.67 6.0 6.0 4 4.89 5.89 7.2 8.0 4 4.89 5.89 7.2 8.0

• Gene Name:

  Glutathione

• Type:

  Application Kit

• Detection Method:

  Fluorometric

• Assay Principle:

  The DetectX® Glutathione Kit is designed to quantitatively measure glutathione (GSH), and oxidized glutathione (GSSG) present in a variety of samples. The kit is unique in that both free and oxidized glutathione are detected in the same well in the microtiter plate. No separation or washing is required. Total glutathione is the sum of GSSG plus GSH. Please read the complete kit insert before performing this assay. A GSH 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 to yield a highly fluorescent product. After mixing the sample or standard with ThioStar® and incubating at room temperature for 15 minutes, the fluorescent product is read at 510 nm in a fluorescent plate reader with excitation at 390 nm. The concentration of the GSH in the sample is calculated, after making a suitable correction for any dilution of the sample, using software available with most fluorescence plate readers. Free glutathione, GSH, is read first after 15 minutes, followed by addition of a reaction mixture that converts all the oxidized glutathione, GSSG, into free GSH, which then reacts with the excess ThioStar® to yield the signal related to Total GSH content. The total concentration of GSH generated in the sample is calculated from the generated signal. We have provided a 96 well plate for measurement 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.

• Assay Protocol:

  Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Reconstitution & Storage Instructions Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Antibody Array (AA) Protocol

• Sample Type:

  Whole Blood, Serum, Plasma, Erythrocytes, Urine, Cell Lysates and Tissue Samples

• Sensitivity:

  Sensitivity was determined as 45 nM in the Free GSH and 48 nM in the Total GSH assays.

• Reconstitution:

  2°C to 8°C

• Components:

  Component Quantity Black Half Area 96 Well Plate 1 or 5 Each Glutathione Standard 100 uL or 300 uL ThioStar® Detection Reagent 2 Plastic vials or 4 Glass vials Dry DMSO 4 mL or 20 mL Assay Buffer Concentrate 35 mL or 200 mL NADPH Concentrate 300 uL or 1.4 mL Glutathione Reductase Concentrate 300 uL or 1.4 mL Oxidized Glutathione Control 300 uL Component Quantity Component Quantity Black Half Area 96 Well Plate 1 or 5 Each Black Half Area 96 Well Plate 1 or 5 Each Glutathione Standard 100 uL or 300 uL Glutathione Standard 100 uL or 300 uL ThioStar® Detection Reagent 2 Plastic vials or 4 Glass vials ThioStar® Detection Reagent 2 Plastic vials or 4 Glass vials Dry DMSO 4 mL or 20 mL Dry DMSO 4 mL or 20 mL Assay Buffer Concentrate 35 mL or 200 mL Assay Buffer Concentrate 35 mL or 200 mL NADPH Concentrate 300 uL or 1.4 mL NADPH Concentrate 300 uL or 1.4 mL Glutathione Reductase Concentrate 300 uL or 1.4 mL Glutathione Reductase Concentrate 300 uL or 1.4 mL Oxidized Glutathione Control 300 uL Oxidized Glutathione Control 300 uL