GSH/GSSG Ratio Assay Kit (Fluorescence)
|
Catalog Number:
CA-G056
|
Detailed Description
|
CA-G056
|
|
Name
|
Elite™ Glutathione GSH/GSSG Ratio Assay Kit (Fluorescence)
|
|
Description
|
This Kit provides an ultrasensitive assay to quantify GSH in the sample. This kit uses a proprietary non-fluorescent dye that becomes strongly fluorescent upon reacting with GSH. With a one-step fluorimetric method, the kit can detect as little as 1 picomole of GSH or GSSG in a 100 μl assay volume.
|
|
Application
|
The assay can be conveniently performed in a 96-well or 384-well microtiter-plate format and readily adapted to automation without a separation step. Its signal can be easily read by a fluorescence microplate reader at Ex/Em = 490/520 nm.
|
|
Size
|
200 assays
|
|
Detection
|
Fluorescence microplate reader
|
|
For Downloading
|
==>> DATA SHEET ==> MSDS
|
Selected Citation:
Patel, D., Mahimainathan, L., Narasimhan, M., Rathinam, M. & Henderson, G. Ethanol (E) Impairs Fetal Brain GSH Homeostasis by Inhibiting Excitatory Amino-Acid Carrier 1 (EAAC1)-Mediated Cysteine Transport. Int. J. Mol. Sci. 18, 2596 (2017).
Frequently Asked Questions and Answers:
1. Q: What would you suggest to prepare the cell samples for this assay?
Answer: All the detergents in various lysis buffers will cause background noise for the assay. The traditional way to do it is the "freeze and thaw" method (i.e. freeze for 20-30 minutes at -80 C then thaw at room temperature, 2-3 times), but it is too tedious. We found that 0.2% SDS gives the best results among all the detergents tested.
2. Q: Does this Kit work for plant samples? Could you recommend a special protocol to measure gluthatione in plants?
Answer: As long as the sampl chop 2 g of plant parts (leaf, root, grain, etc.) and ground it in a homogenizer for 2 min with 5-10 ml of 0.1 N HCl; centrifuge to pbtain the GSH containing supernatant, then adjust pH to 6.5 for the GSH assay.
3. Q: My sample is frozen brain tissue, how should I prepare it for the saasy?
Answer: It is better use PBS, and simply homogenize it, then use the supernatant.
4. Q: Does this kit measure GSSG and GSH directly?
Answer: This kit can be used for measuring total and the reduced form (GSH) directly, the oxidized form (GSSG) is calculated [the total minus GSH]. The GSSG probe is a reducing agent that reduces GSSG to GSH (in order to measure total GSH). The detection limit is around 0.1~0.3 uM.
5. Q: I work with human primary cells. How many cells should I use in the assay?
Answer: Suggest to start with 10,000 cells/well first.
6. Q: Is any of the components in this kit from animals?
Answer: No. The only enzyme (in Component E) is from Baker's Yeast (S. cerevisiae).
7. Q: For the fluorescent detection, an emission filter with 520 nm is recommended. But we have only 535 nm filter. Would it work for this measurement?
Answer: Yes.
8. Q: We don't have the filters for 490/520 (nm). What we have are for 395/535 (nm). Would it work?
Answer: No. the emission is OK, but the excitation is not. The lowest excitation wavelength is at 460 nm.
9. Q: I've tried this kit, but it didn't well for my samples. Here is the protocol I used for obtaining cell culture lysates. Could you take a look and give your suggestion?
Answer:
- remove supernatant, then wash with HEPES-BSS;[Fine] - trypsinize for 2-6 min, stop trypsination;[We suggest to avoid trypsin and try using a rubber scrapper or EDTA to harvest cells, then centrifuge the cells and use the supernatant] - centrifuge pellet down at 180g, 5min, 4°C, then wash with 5ml cold PBS; count cells; [Fine] - centrifuge pellet down at 180g, 5min, 4°C; [May need to use more cells since you indicated the signal in the undiluted sample is very low.] - wash with 5ml COLD PBS; resuspend in ice-cold 5% MPA;[Fine. 5% MPA could be added to the supernatant for deproteination.] - mix thoroughly, homogenize by sonication (2x 5 cycles), then centrifuge at 12.00rpm for 5min at 4°C;[Fine] - collect supernatant for GSH assay; store on ice OR at -80°C. [Need to neutralize the MPA to pH 4~6, then analyze the undiluted sample with the kit]
|
|
|