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Amplite® Fluorimetric Glutathione Assay Kit *Green Fluorescence* | 10055

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SKU:
AB-10055-GEN
Availability:
IN STOCK
NULL167.00

Description

Amplite® Fluorimetric Glutathione Assay Kit *Green Fluorescence* | 10055

The monitoring of reduced and oxidized glutathione (GSH) in biological samples is essential for evaluating the redox and detoxification status of cells and tissues in relation to the protective role of glutathione against oxidative and free-radical-mediated cell injury. Cysteine metabolism disorders include cystinosis, an autosomal recessive disease produced by a defect in lysosomal transport, and cystinuria, a common heritable disorder of amino acid transport. Cysteine is unique among the amino acids found in proteins. There are a few reagents or assay kits available for quantitating thiols in biological systems. However, all the commercial kits either lack sensitivity or have tedious protocols. Our Amplite® Fluorimetric Glutathione Qutitation Kit provides an ultrasensitive fluorimetric assay to quantitate GSH in sample. The kit uses a proprietary non-fluorescent dye that becomes strongly fluorescent upon reacting with thiol. The kit provides a sensitive, one-step fluorimetric method to detect as little as 1 picomole of cysteine or GSH in a 100 µL assay volume. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation without a separation step. Its signal can be easily read using a fluorescence microplate reader.

Platform


Fluorescence microplate reader

Excitation 490 nm
Emission 525 nm
Cutoff 515 nm
Recommended plate Solid black

Components


Component A: Thiolite™ Green 1 vial
Component B: Assay Buffer 1 bottle (25 mL)
Component C: GSH Standard 1 vial (62 µg)
Component D: DMSO 1 vial (200 µL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare GSH working solution (50 µL)
  2. Add GSH standards or test samples (50 µL)
  3. Incubate at RT for 10 to 60 minutes
  4. Monitor the fluorescence increase at Ex/Em = 490/525 nm (Cutoff = 515 nm)

Important notes
Thaw all the kit components at room temperature before starting the experiment.

 

PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. GSH standard solution (1 mM):
Add 200 µL of Assay Buffer (Component B) into the vial of GSH Standard (Component C) to make 1 mM (1 nmol/µL) GSH standard solution.

2. Thiolite™ Green stock solution (100X):
Add 100 µL of DMSO (Component D) into the vial of Thiolite™ Green (Component A) to make 100X Thiolite™ Green stock solution.

PREPARATION OF STANDARD SOLUTION

GSH standard

For convenience, use the Serial Dilution Planner: 

Add 30 μL of 1 mM (1 nmol/µL) GSH standard solution to 970 μL of Assay Buffer (Component B) to generate 30 μM (30 pmol/µL) GSH standard solution. Take 30 μM (30 pmol/µL) GSH standard solution and perform 1:3 serial dilutions to get serially diluted GSH standards (SD7-SD1) with Assay Buffer (Component B). Note: Diluted GSH standard solution is unstable. Use within 4 hours.

 

PREPARATION OF WORKING SOLUTION

Add 50 μL of 100X Thiolite™ Green stock solution into 5 mL of Assay Buffer (Component B) and mix well to make GSH working solution. 

For guidelines on cell sample preparation, please visit

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Additional Information

Size:
200 TESTS
Storage:
-20°C
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