GSH/GSSG Assay Kit

Catalog No. ABIN1000314

Product Details

Target: GSH/GSSG

Application: Biochemical Assay (BCA)

Sample Type: Blood, Cell Culture Supernatant, Cell Extracts, Plasma, Serum, Urine

Specificity: 0.10 nM

Characteristics: Sensitive and accurate. Linear detection range 0.01-3 µM GSH equivalents with a detection limit of 10 nM GSH equivalents.

Components: Scavenger: 500 µL. NADPH: 40 µL. DTNB: 60 µL. 2X Assay Buffer: 25 mL. GR Enzyme: 120 µL. Glutathione Standard: 50 µL.

Material not included: Pipetting devices, clear flat-bottom 96-well plates, plate reader capable of reading optical density at 412 nm, centrifuge tubes and table centrifuge.

Application Details

Application Notes: Direct Assays: total, reduced and oxidized glutathione in whole blood, plasma, serum, urine, tissue and cell extracts.
Drug Discovery/Pharmacology: effects of drugs on glutathione metabolism.

Protocol: Important: equilibrate Scavenger, DTNB and 2X Assay Buffer to room temperature before use. Dilute 2X Assay buffer with an equal volume of dH2O to make 1X Assay Buffer. Briefly mix GR Enzyme before use. Note: mercaptoethanol, dithiothreitol and cysteine are known to interfere in this assay. Avoid using these compounds during sample preparation.
1. Standards. First dilute GSH standard to 300 µMby mixing 3 µL 100 mM Standard with 997 µL dH2O. Next, prepare the 3 µMPremix by mixing 5 µL of the 300 µMGSH with 495 µL 1X Assay Buffer. Dilute standards in1.5-mL centrifuge tubes as described in the Table. Transfer 200 µL of each Standard to separate wells in a 96 well plate.
2. Glutathione Detection. Prepare enough working reagent (WR) for 4 standards and all samples. For each reaction combine the following: 105 µL 1X Assay Buffer, 1 µL GR Enzyme, 0.25 µL NADPH and 0.5 µL DTNB. Mix WR immediately after adding the DTNB. Add 100 µL of WR to each Standard and Sample well. Mix well.
3. Read OD412nm at 0 min and again at 10 min.

Sample Preparation:

Sample Preparation for GSSG Measurement Cell lysate can be prepared : wash cells (1-2 x 10 6 ) in cold PBS. Lyse cells by homogenization or sonication in 200 µL of cold buffer containing 50 mM phosphate (pH = 7), 1 mM EDTA, and 20 µL Scavenger. Centrifuge at 10,000g for 5 min at 4°C. Transfer supernatant to a clean tube and proceed to the deproteination procedure. Whole blood samples can be prepared : mix 50 µL whole blood with 5 µL Scavenger and freeze at -70°C. (Freezing helps lyse the blood cells). After freezing, thaw and mix sample. Incubate at RT for 2-10 min then proceed to the deproteination procedure.

Sample Preparation for Total Glutathione Measurement Cell lysate can be prepared : wash cells (1-2 x 10 6 ) in cold PBS. Lyse cells by homogenization or sonication in 1 mL of cold buffer containing 50 mM phosphate (pH = 7) and 1 mM EDTA. Centrifuge at 10,000g for 15 min at 4°C. Transfer supernatant to a clean tube and proceed to the deproteination procedure. Whole blood samples can be prepared : freeze 50 µL whole blood at -70°C. (Freezing helps lyse the blood cells). After freezing, thaw and mix sample. Incubate at RT for 2-10 min then proceed to the deproteination procedure. Deproteination Procedure. Prepare a solution of 5wt% Metaphosphoric Acid (available separately at BioAssay Systems under cat# MPA-2G) in water (MPA Reagent). This reagent must be prepared fresh daily. Add 65 µL MPA Reagent to 25 µL sample, briefly vortex to mix and then centrifuge at 14000 rpm for 5 min. For total glutathione whole blood samples, transfer 5 µL of clear supernatant to a clean tube and mix with 620 µL 1X Assay Buffer. For all other samples, transfer 6 µL of clear supernatant to a clean tube and mix with 244 µL 1X Assay Buffer. Transfer 200 µL of each neutralized deproteinated sample to separate wells of a 96 well plate.

Restrictions: For Research Use only

Handling

Storage: -20 °C

References

Pampliega, Domercq, Soria, Villoslada, Rodríguez-Antigüedad, Matute: "Increased expression of cystine/glutamate antiporter in multiple sclerosis." in: Journal of neuroinflammation, Vol. 8, pp. 63, (2011) (PubMed).

Target Details

Target: GSH/GSSG

Background: Quantitative determination of total, reduced (GSH) and oxidized (GSSG) glutathione by colorimetric (412nm) method.
Procedure: 20 min.

Glutathione, a tripeptide of glycine, glutamic acid and cysteine, is one of the key antioxidants involved in protecting cells from damages by reactive oxygen species. Glutathione (GSH) reduces disulfide bonds in cytoplasmic proteins to cysteines, in which it is converted to its oxidized form GSSG. This GSH/GSSG Assay Kit is designed to accurately measure total, reduced and oxidized glutathione in biological samples using an enzymatic method that utilizes Ellman's Reagent (DTNB) and glutathione reductase (GR). DTNB reacts with reduced glutathione to form a yellow product. The rate of change in the optical density, measured at 412 nm, is directly proportional to glutathione concentration in the sample. This kit can also be used to measure oxidized (GSSG) by using a specific protocol which first scavenges all GSH with 1-methyl-2-vinylpyridinium triflate.