|Product Includes||Quantity (with Count)||Storage Temp|
|Reduced Glutathione Standard||1 x 1 ea||-20°C|
|Glutathione-S-Transferase||1 x 1 ea||-20°C|
|Tris Assay Buffer||1 x 25 ml||+4°C|
|Digitonin Lysis Buffer||1 x 11 ml||-20°C|
|Monochlorobimane||1 x 1 ea||-20°C|
Upon receipt, Tris Assay Buffer (#13865) should be removed from #13859 and stored at 4°C. And remaining components should be stored at -20ºC.
Reduced Glutathione Working Solution as follows (see Table 1).
Table 1: GSH Working Solution for one 96-well plate (100 assays)
|Kit Component||Volume (µl)|
|Tris Assay Buffer||4930|
NOTE: To create a GSH standard curve, prepare a 100 µM GSH Standard Solution by diluting the 100 mM Reduced Glutathione Standard 1:1000 in Tris Assay Buffer. Run the standard curve in triplicate using 50 µl of standard per well. Create a five point standard curve by starting at 100 µM and using a 1:2 serial dilution in Tris Assay Buffer. Include a blank control. The GSH Standard Solution is not stable at this concentration and should be used within 2 hours or discarded.
Additional Reagents (not supplied): DMSO ( #12611)
NOTE: If using suspended or detached cells, collect and centrifuge the cell suspension for 5 min at 1,200 rpm. Discard the supernatant and add Digitonin Lysis Buffer to the cell pellet.
NOTE: use a 10 kDa MWCO centrifugal filter and follow the manufacturer’s instructions for cells that require an additional filtration step.
Table 2: Detection and Standard Curve Assays
|Sample||Test Sample (µl)||GSH Standard (µl)||Working Solution (µl)||Assay Buffer (µl)||Total Volume (µl)|
|Sample||1-50||-||50||50 minus sample volume||100|
The following procedure measures reduced glutathione in live cells using a 96-well plate. The same protocol can be modified for fluorescent imaging and flow cytometry detection.
NOTE: For best results, a cell number titration is recommended to determine the optimal cell seeding density. We recommend not exceeding 1x105 cells per well.
posted June 2020
Protocol Id: 2050
|CAS||77-86-1, 57-50-1, 76421-73-3|
All Species Expected
Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.
Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST's products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST's Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.