Product Details
| abID | ab239709 |
|---|---|
| Cat # +Size | K261-100 |
| Size | 100 assays |
| Detection Method | Absorbance (412 nm) |
| Species Reactivity | Mammalian |
| Applications | The Colorimetric Glutathione Detection Kit provides a simple in vitro assay for detecting the GSH changes in apoptosis and other pathological processes. |
| Features & Benefits | • Simple procedure; takes only ~2-3 hours • Fast and convenient |
| Kit Components | • Glutathione Reaction Buffer • Glutathione Substrate (DTNB) • NADPH Generating Mix (lyophilized) • Glutathione Reductase (lyophilized) • Sulfosalicylic Acid (SSA, 1 gram) • GSH Standard (lyophilized, MW 307) |
| Storage Conditions | -20°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
Glutathione (GSH) is the major intracellular low-molecular-weight thiol that plays a critical role in the cellular defense against oxidative stress in mammalian cells. BioVision’s ApoGSH™ Glutathione Colorimetric Assay Kit provides a convenient, colorimetric method for analyzing either total glutathione or the reduced form glutathione alone using a microtiter plate reader. The assay is based on the glutathione recycling system by DTNB and glutathione reductase (Fig. 1). DTNB and glutathione (GSH) react to generate 2-nitro-5-thiobenzoic acid which has yellow color. Therefore, GSH concentration can be determined by measuring absorbance at 412 nm. The generated GSSG can be reduced back to GSH by glutathione reductase, and GSH reacts with DTNB again to produce more 2-nitro-5-thiobenzoic acid. Therefore, the recycling system dramatically improves the sensitivity of total glutathione detection. The kit includes the 5-Sulfosalicylic acid (SSA) for the removal of proteins from samples and for the protection of GSH oxidation and γ-glutamyl transpeptidase reaction. The kit can quantify glutathione from 1-100 ng/well in a 200 l reaction. For detecting lower glutathione concentrations, such as in blood samples, increasing reaction time will generate stronger signal. The kit can also specifically detect the reduced form of glutathione (GSH) by omitting the glutathione reductase from the reaction mixture. The sensitivity for detecting the reduced form of glutathione (without recycling system) is 100 times lower than detecting the total glutahione.
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What are the differences between K251 &K261?
1) K251 cannot measure total glutathione. It measures the relative level of glutathione between untreated and treated samples.
2) You cannot compare the sensitivity between K251 and K261, as K261 measure absolute amount, whereas K251 measures relative fluorescence level between samples.
3) K261 cannot measure GSSG. You need to use K264 to measure GSH, GSSG, and total amount, individually.
2) You cannot compare the sensitivity between K251 and K261, as K261 measure absolute amount, whereas K251 measures relative fluorescence level between samples.
3) K261 cannot measure GSSG. You need to use K264 to measure GSH, GSSG, and total amount, individually.
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 frozen samples with this assay?
Fresh samples are always preferred over frozen samples. However, frozen samples can also be used, provided, they were frozen right after isolation, were not freeze thawed multiple time (for which we recommend aliquoting the samples before freezing) and have been frozen for relatively short periods.
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.
How do I normalize my samples against protein concentration
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.
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.
Can RIPA buffer be used to lyse the cells?
We do not recommend using the RIPA buffer for sample preparation. Please use glutathione reaction buffer provided in the kit for sample preparation.
Can I use serum samples with this kit?
Yes, you may use serum samples. However, serum may have low levels of glutathione.
Does this kit measure total glutathione or the reduced form of glutathione in cell lysates? Can I analyze glutathione in the mitochondrial fraction using this kit? I purchased, Catalogue # K288 from Biovision - “Mammalian Mitochondria Isolation Kit For Tissue & Cultured Cells” and will this kit be compatible with K261?
K261 measures both total glutathione and reduced glutathione. Please omit Glutathione Reductase from the reaction mixture when you measure reduced glutathione in your samples as mentioned in the data sheet. Yes, you can certainly use purified mitochondria as a sample to measure glutathione. Please start with 50-100 µg of mitochondrial protein. Please isolate the mitochondria and then lyse it with K261-100-1, Glutathione Reaction Buffer and always work with freshly isolated mitochondria.
Jingsi Zhang, et al., (2018) Jia-Jian-Di-Huang-Yin-Zi decoction exerts neuroprotective effects on dopaminergic neurons and their microenvironment, Scientific Reports, Jun. 2018, 29959371
Park, S. et al., (2017) Proteasome inhibitor-induced cleavage of HSP90 is mediated by ROS generation and caspase 10-activation in human leukemic cells, Redox Biology, Oct.2017, 13: 470-476
Wang, Y. et al., (2017) Quercetin and cyanidin-3-glucoside protect against photooxidation and photodegradation of A2E in retinal pigment epithelial cells, Experimental Eye Research, Jul.2017, 160:45-55
Yuan, CW et al., (2017) Evaluation of efficacy and toxicity of exfoliated silicate nanoclays as a feed additive for fumonisin detoxification, J. Agric. Food Chem, 2017, Just accepted
Roh, Jong-Lyel et al. (2017) Nrf2 inhibition reverses the resistance of cisplatin-resistant head and neck cancer cells to artesunate-induced ferroptosis, Redox Biol. 2017 Apr;11:254-262.
For more citations of this product click here
