Description
GSH/GSSG Assay Kit (BA0118) (BA0118)
The GSH/GSSG Assay Kit (SKU: BA0118) allows accurate measurement of total, reduced and oxidised glutathione in biological samples. Glutathione is a tripeptide that is one of the key antioxidants protecting cells from reactive oxygen species; reduced glutathione (GSH) is converted to its oxidised form (GSSG) as it reduces disulphide bonds. The assay uses an enzymatic method with Ellman's Reagent (DTNB) and glutathione reductase (GR): DTNB reacts with reduced glutathione to form a yellow product, and the rate of change in optical density at 412 nm is directly proportional to the glutathione concentration. Oxidised glutathione is measured using a protocol that first scavenges all GSH.
| Product Name: | GSH/GSSG Assay Kit (BA0118) |
| SKU: | BA0118 |
| Detection Method: | Colorimetric (412 nm) |
| Detection Range: | 0.01 - 3 uM GSH equivalents (detection limit 10 nM) |
| Sample Type: | Whole blood, plasma, serum, urine, tissue and cell extracts |
| Species Reactivity: | All |
| Assay Time: | 10 minutes |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
A colorimetric assay for total, reduced and oxidised glutathione using a DTNB/GR recycling method with a deproteination step.
- Sensitive and accurate with a linear detection range of 0.01 - 3 uM GSH equivalents and a detection limit of 10 nM
- Direct assay of total, reduced and oxidised glutathione in whole blood, plasma, serum, urine, tissue and cell extracts
- Studying the effects of drugs on glutathione metabolism
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Equilibrate Scavenger, DTNB and 2x Assay Buffer to room temperature. Dilute the 2x Assay Buffer with an equal volume of distilled water to make 1x Assay Buffer, and briefly mix the GR Enzyme. |
| 2 | Sample preparation for GSSG: lyse cells or whole blood in cold buffer containing the Scavenger to remove GSH, then proceed to deproteination. For total glutathione, prepare samples without Scavenger. |
| 3 | Deproteination: prepare 5% metaphosphoric acid solution fresh, add 65 uL to 25 uL sample, vortex and centrifuge at 14,000 rpm for 5 min, then neutralise the supernatant with 1x Assay Buffer as directed. Transfer 200 uL of each neutralised sample to a 96-well plate. |
| 4 | Standards: dilute GSH standard to 300 uM then prepare a 3 uM Premix and dilute per the table. Transfer 200 uL of each standard to separate wells. |
| 5 | Glutathione Detection: prepare Working Reagent per reaction from 105 uL 1x Assay Buffer, 1 uL GR Enzyme, 0.25 uL NADPH and 0.5 uL DTNB. Add 100 uL to each standard and sample well and mix. |
| 6 | Read OD412nm at 0 minutes and again at 10 minutes. |
Subtract OD0min from OD10min for each standard and sample, then subtract the delta-OD blank (standard 4) and plot delta-delta-OD against concentration to obtain the slope. [GSH total] = (delta-ODSAMPLE - delta-ODBLANK) / Slope x n (uM); [GSSG] = 0.5 x (delta-ODS(GSSG) - delta-ODBLANK) / Slope x n (uM); [GSH] = [GSH total] - 2 x [GSSG] (uM). The dilution factor n is 165 for Scavenger-treated samples, 450 for untreated whole blood and 150 for other untreated samples. 1 mg/dL glutathione equals 32.5 uM.
| Component | Quantity | Storage |
| Scavenger | 500 uL | -20C |
| NADPH | 40 uL | -20C |
| DTNB | 60 uL | -20C |
| Assay Buffer (2x) | 25 mL | -20C |
| GR Enzyme | 120 uL | -20C |
| Glutathione Standard | 50 uL | -20C |
