Product Details
| Cat # +Size | K264-100 |
|---|---|
| Size | 100 assays |
| Detection Method | Fluorescence (Ex/Em 340/420 nm) |
| Species Reactivity | Mammalian |
| Applications | The kit provides unique formula and buffers and procedures for detecting the GSH, GSSG, and total glutathione individually |
| Features & Benefits | • Simple procedure; takes only ~1-2 hours • Fast and convenient • The assay is easy to perform and detects 2-400 ng/µl of GSH, GSSG or total glutathione. |
| Kit Components | • Glutathione Assay Buffer • PCA (Perchloric Acid, 6N) • KOH (<6N) • OPA Probe (o-phthalaldehyde ) • Reducing Agent Mix • GSH Quencher • GSH Standard (FW: 307) |
| Storage Conditions | -20°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
Glutathione is the major intracellular low-molecular-weight thiol that plays a critical role in cellular defense against oxidative stress in tissues and cells. Commercially available glutathione detection kits, such as the DTNB-enzyme cycling glutathione assay kit or the Monochlorobimane based assay kit hardly distinguish between reduced glutathione (GSH; FW: 307) and oxidized glutathione (GSSG; FW: 612). BioVision’s Glutathione Detection Kit provides a unique, convenient tool for detecting GSH, GSSG, and total glutathione individually. In the assay, OPA, reacts with GSH (not GSSG), generating fluorescence, so GSH can be specifically quantified. Adding a reducing agent converts GSSG to GSH, so (GSH + GSSG) can be determined. To measure GSSG specifically, a GSH Quencher is added to remove GSH, preventing reaction with OPA (while GSSG is unaffected). Reducing agent is then added to destroy excess quencher and to convert GSSG to GSH. Thus, GSSG can be specifically quantified. The kit provides a unique procedure and buffer formula to eliminate protein thiol interference and to stabilize GSH and GSSG in solution.
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My GSSG is higher than GSH. What can I do to improve the ratio?
The GSH:GSSG in a healthy cell would be around 20:1. Under certain conditions, GSH is converted to GSSG very rapidly in the presence of enzymes. We suggest treating samples with PCA immediately in order to stop the conversion as mentioned in the data sheet. The samples, right from the extracting the cells from the cell culture plate itself, have to be processed on ice to minimize any impact on the GSH and GSSG as they are both very labile. Within cells GSH is 300 - 800 folds higher than GSSG. Therefore, even a minimal oxidation of GSH can induce a dramatic increase of GSSG. Therefore, we suggest deproteinizing the samples immediately after collection followed by the addition of quencher for GSSG samples. This will help to minimize the GSH to GSSG coversion.
Can we use frozen samples with this assay?
We highly recommend using fresh samples with this assay. Under certain conditions GSH conversion to GSSG is very rapid and therefore the results can get skewed towards GSSG. In case of use of frozen samples, please ensure that the samples are frozen right after isolation/preparation and have not undergone multiple freeze thaw cycles.
The detection limit according to the user manual is 2-400 ng/ul. How much is it in uM?
The molecular weight of GSSG is 612 Da. So the 2-400ng/ul will be 3.3-650 uM.
The GSH:GSSGin a healthy cell would be around 20:1. That means the amount of GSSG in a cell is significantly lower than GSH. Could BioVisions kit still have sensitivity for detecting GSSG without the interference by the GSH or other factors? Do the users need to be careful about any other things to generate the best results?
Measuring GSSG separately from GSH is the unique advantage of K264-100 kit. In BioVisions assay protocol, GSH in samples can be specifically quenched before assaying for GSSG. Therefore GSSG can be specifically measured without any interference from GSH. The kit has a straightforward principle, is very easy to use, and repeatable.
I am using your Glutathione assay kit to measure GSH, GSSG, and total glutathione in red blood cells. I understand that this kit will eliminate interference from protein thiols but I have been reading that protein sulfhydryls can be an important reservoir in the GSH/GSSG system (Di Simplicio et al. 1998 Arch. Biochem. Biophys). Is there any way I can keep the precipitated proteins, liberate any glutathione, and use them in a different assay?
Under certain conditions, GSH is converted to GSSG very rapidly in the presence of enzymes. The half life of GSH is a few minutes if not seconds. That is the nature of the material. We suggest that you treat your sample with PCA as soon as you can in order to stop the conversion as mentioned in the data sheet. There are no enzymes in the standard, so the pure standard alone is relatively stable.
Can this kit be used with samples like bacteria, plants, drosophila etc?
We have optimized the kit with mammalian samples. However, theoretically these kits should work with samples from multiple species/sources. Since the optimal conditions depend on the sample type, the protocol has to be be adapted to fit the samples for efficient results. Please refer to this kit's citations to see what kind of samples have been used with this kit other than mammalian samples.
Can we use a different wavelength than recommended for the final analysis?
It is always recommended to use the exact recommended wavelength for the most efficient results. However, most plate readers have flexibility in their band width of detection in increments of +/- 10 nm. Depending on this flexibility range, you can deviate from the recommended wavelengths within limits.
What is the exact volume of sample required for this assay?
There is no specific volume we can recommend for the amount any sample to be used since it is completely sample concentration and quality based. You have to do a pilot expt with multiple sample volumes to determine the optimal volume which gives a reading within the linear range of the standard curve. Please refer to the citations for this product to see what other clients have used with similar sample types.
Do you have trial sizes of this kit?
Unfortunately, we do not have trial sizes of this kit available. However, if you are based in the US or Canada, we will give you a 10% off list price introductory discount on its purchase price. If you are based out of this area please contact yopur regional BioVision distributor.
What is the shelf life of this kit?
This kit is good for 12 months from the date of shipment in the unopened form when stored at the appropriate temperature and appropriate conditions. After opening and reconstitution, some of the components in this kit are good for 2 months at -20C. Please refer to the datasheet for storage information and shelf life of each of the components.
Why are my standard curve values lower than those shown on the datasheet?
There are multiple factors which influence the signals like the incubation times, room temperature, handling etc. In general, to increase the value of the standards, you can increase the incubation time. As long as the standard curve is linear, it should be fine to use, since all of your samples will also be measured under the same conditions on this curve.
You can use a protein quantitation assay on the supernatants you get from cell/tissue lysates or with any other liquid sample in the assay buffer.
Can we purchase individual components of this kit?
Yes, you can purchase any of the kit's components without the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Why did I obtain higher value for reduced glutathione than total glutathione?
When detecting the reduced form of glutathione, protein thiols can generate significant background signal. In such cases, it is necessary to completely remove proteins from samples. We suggest using Amicon Centrifugal Spin column with 10K molecular weight cut off filter to remove proteins. Then the reduced glutathione can be easily detected from spin through samples.
Can we use an alternate buffer for sample preparation (cell lysis, sample dilutions etc)?
Our assay buffers are optimized for the reactions they are designed for. They not only contain some detergents for efficient lysis of your cells/tissue, but also contain some proprietary components required for the further reactions. Therefore, we highly recommend using the buffers provided in the kit for the best results.
Should I make a standard curve for every expt I do, or is one curve/kit enough?
Yes, I would strongly recommend you to do the standards every time you do the expt. There is always a chance that something was done differently that day and we do not want any conditions to differ between standards and samples.
Lee, Geum-Hwa et al. (2017) Protective effect of Curcuma longa L. extract on CCl4-induced acute hepatic stress, BMC Res Notes. 2017 Feb 1;10(1):77.
Charitou et al., FOXOs support the metabolic requirements of normal and tumor cells by promoting IDH1 expression. EMBO Rep., Apr 2015; 16: 456 - 466.
Odkhuu et al., Involvement of redox balance in in vitro osteoclast formation of RAW 264.7 macrophage cells in response to LPS.Innate Immunity, Feb 2015; 21: 194 - 202.
Ishaq et al., Atmospheric gas plasma–induced ROS production activates TNF-ASK1 pathway for the induction of melanoma cancer cell apoptosis. Mol. Biol. Cell, May 2014; 25: 1523 - 1531.
Chowdhury et al., Disuccinyl Betulin Triggers Metacaspase-Dependent Endonuclease G-Mediated Cell Death in Unicellular Protozoan Parasite Leishmania donovani. Antimicrob. Agents Chemother., Apr 2014; 58: 2186 - 2201.
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