Product Details
| abID | ab65322 |
|---|---|
| Cat # +Size | K251-100 |
| Size | 100 assays |
| Detection Method | Fluorescence (Ex/Em 380/461 nm) |
| Species Reactivity | Mammalian |
| Applications | The Glutathione Detection Kit provides a simple in vitro assay for detecting the GSH changes in apoptosis and other pathological processes. |
| Features & Benefits | • Simple two-step procedure; takes only 2 hours • Fast and convenient • The assay utilizes a dye that has a high affinity for glutathione. The unbound dye is almost nonfluorescent; whereas the dye fluoresces blue when bound to glutathione. |
| Kit Components | • Cell Lysis Buffer • Monochlorobimane • GST Reagent • GSH Standard (1 mg; MW: 307) |
| Storage Conditions | -20°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
Glutathione is the principal intracellular low-molecular-weight thiol that plays a critical role in the cellular defense against oxidative and nitrosative stress in mammalian cells. Diminished glutathione levels have been observed in the early stages of apoptosis. BioVision’s ApoGSH™ Glutathione Detection Kit provides a simple in vitro assay for detection of total glutathione changes in apoptosis and other samples. The assay utilizes monochlorobimane (MCB), a dye that appears to form an adduct exclusively with glutathione. The unbound MCB is almost nonfluorescent, whereas the dye fluoresces blue (Ex./Em. = 380nm/461nm) when bound to glutathione of reduced or oxidized form. The reaction is catalyzed by glutathione S-transferase. Thus, the amount of total glutathione can be easily detected using a fluorometer or a 96-well fluorometric plate reader.
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The assay uses the dye monochlorobimane (MCB), which forms an adduct with glutathione in a reaction catalyzed by glutathione-S-transferase (GST). The oxidized glutathione should not react with MCB, so this kit doesn't detect oxidized glutathione. Therefore, the description in first paragraph “The assay detects both reduced and oxidized glutathione.” may be wrong. Could you explain how the kit can detect oxidized glutathione?
The kit detects reduced Glutathione (GSH) as MCB forms an adduct with GSH. However, a reducing agent is added in the buffer, which will convert the oxidized glutathione (GSSG) to GSH. Therefore, the kit cannot detect oxidized and reduced glutathione separately and detects total glutathione (GSH and GSSG converted to GSH).
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?
AUnfortunately, 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 -20°C. 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.
Isabelle K. Vila, A muscle-specific UBE2O/AMPKα2 axis promotes insulin resistance and metabolic syndrome in obesity. JCI Insight, July 2019; 31292296.
Suresh, S., et al., (2017) , Broccoli (Brassica oleracea) Reduces Oxidative Damage to Pancreatic Tissue and Combats Hyperglycaemia in Diabetic Rats, Prev Nutr Food Sci., 2017, 22(4): 277–284.
Waly et al., Low nourishment of B-vitamins is associated with hyperhomocysteinemia and oxidative stress in newly diagnosed cardiac patients. Experimental Biology and Medicine, Aug 2015; 10.1177/1535370215596860.
Al-Numair, A. S. et. al. Dietary folate protects against azoxymethane-induced aberrant crypt foci development and oxidative stress in rat colon. Exp Biol Med, Sep 2011; 236: 1005 - 1011.
Guha, R. et. al. Intracellular GSH Depletion Triggered Mitochondrial Bax Translocation to Accomplish Resveratrol-Induced Apoptosis in the U937 Cell Line. J. Pharmacol. Exp. Ther., Jan 2011; 336: 206 - 214.
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