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Index / Trevigen / HT Glutathione Assay Kit / Product Detail : 7511-100-K HT Glutathione Assay Kit
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#7511-100-K HT Glutathione Assay Kit

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  Price : 392   EUR
445   USD
304   GBP
1649   Zloty
52532   JPY
3027   NOK
3244   SEK
444   CHF

Product name : HT Glutathione Assay Kit

Catalog number : 7511-100-K

Quantity: 384 tests

Availability: Yes

Supplier name : Trevigen

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More Details about


For Research Use Only. Not For Use In Diagnostic Procedures


HT Glutathione Assay Kit


Colorimetric assay for total, reduced and oxidized glutathione. Sufficient reagents for 384 tests.

I. Background


Increased oxidative damage of biomolecules (proteins, lipids and DNA) by free radicals is one of the pathogenic mechanisms of diseases such as cancer, atherosclerosis, inflammation, and neurodegenerative disorders (1, 2). Glutathione, the major intracellular non-protein thiol, is an important protector against free radical damage by providing reducing equivalents for several key antioxidant enzymes and also by scavenging hydroxyl radicals and nascent oxygen. High reduced Glutathione levels are associated with fewer numbers of illnesses, higher levels of self-rated health, lower cholesterol, lower body mass index, and lower blood pressures among the elderly (3). Glutathione provides a primary defense system for the removal of oxidants in the brain. Studies reveal a correlation between low Glutathione levels and damage to neurons that manufacture dopamine, suggesting a link to Parkinson's disease (4). The concentration of Glutathione ranges from 1-10 mM in cells and is in the micromolar range in plasma.


Features of the Kit:


-Suitable for mammalian cells, tissue, blood, plasma and other bodily fluids.
-Contains sufficient reagents to assay 384 data points or to determine Glutathione in:
A) 123 experimental samples, each performed in triplicate, plus one Glutathione standard curve;
B) 108 experimental samples, each performed in triplicate, plus 4 Glutathione standard curves;
C) 88 experimental samples, each performed in triplicate, plus 8 Glutathione standard curves.

II. Principle of the Assay


 HT Glutathione Assay Kit utilizes a carefully optimized enzymatic recycling method for the quantification of glutathione. Glutathione Reductase reduces oxidized glutathione (GSSG) to reduced glutathione (GSH). As shown in Figure 1 (below), the sulfhydryl group of GSH reacts with DTNB (5,5’-dithiobis-2- nitrobenzoic acid, Ellman’s reagent) to produce a yellow colored 5-thio-2- nitrobenzoic acid (TNB) that absorbs at 405 or 414 nm, and the mixed disulfide, GSTNB, that is reduced by Glutathione Reductase to recycle the Glutathione and produce more TNB. The rate of TNB production is directly proportional to this recycling reaction which is in turn directly proportional to the concentration of glutathione in the sample. The measurement of the absorbance of TNB at 405 or 414 nm provides an accurate estimation of glutathione in the sample.


III. Precautions and Limitations


A. For research use only. Not for use in diagnostic procedures.
B. The physical, chemical and toxicological properties of the kit components have not been fully investigated; therefore, Trevigen recommends the use of gloves, lab coats, and eye protection while using these chemical reagents. Trevigen assumes no liability for damage resulting from handling or contact with these products. MSDS are available on request.


 

IV. Materials Supplied


 

V. Reagents/Equipment Required But Not Supplied


Equipment Reagents
-96 well plate reader High quality,
-double-distilled H2O
-Multichannel pipetor Metaphosphoric acid (Aldrich Cat# 23,927-5)
-Pipetor 4-Vinylpyridine (Aldrich Cat# V3204-5ML)
-Pipette tips Reagent alcohol
-Centrifuge (for cell lysis)


VI. Reagent Preparation


1. 1X Assay Buffer
Prior to each experiment, prepare the necessary amount of 1X Assay Buffer by diluting the 25X Assay Buffer (Cat# 7511-100-02) with dH2O.
2. Reaction Mix
Reconstitute one or more bottles of Reaction Mix (Cat # 7511-100-04) with 8 ml of dH2O per bottle. Periodically swirl the bottle gently over a 15 minute period to dissolve contents. Immediately before use in the assay, vortex the vial of Glutathione Reductase (Cat# 7511-100-01) and add 10 μl to the bottle of Reaction Mix. Each bottle of Reaction Mix is sufficient for 53 wells in a 96-well plate, or little more than half a plate. Pool the reconstituted
Reaction Mix together into one tube if more than one bottle is used.
3. 5% (w/v) Metaphosphoric acid
Prepare 5% (w/v) Metaphosphoric acid (Aldrich Cat# 23,927-5, not provided) in dH2O.
4. GSSG The GSSG (Cat# 7511-100-06) is provided at a concentration of 4 μM (4 pmoles/μl). Store any unused portion at 4˚C.
5. 2M 4-Vinylpyridine
This reagent blocks free thiols present in the reaction, thus eliminating any contribution to the cycling reaction caused by GSH. Prepare 2M 4- vinylpyridine solution (Aldrich Cat# V3204-5ML, not provided) by mixing 108 μl 4-vinylpyridine with 392 μl ethanol (solution should be prepared and subsequently used only in a chemical fume hood). Use immediately and discard any unused portion.
Note: It is recommended that you use 4- vinylpyridine within 1 month of purchase and store at -20°C.

VII. Sample Preparation


All samples are treated with 5% (w/v) Metaphosphoric acid to remove proteins which interfere with the assay.
A. Cell Lysate Preparation
1. Detach adherent cells by gentle trypsinization. Count the cells and centrifuge at 300 x g for 10 minutes at 4˚C. Wash the cells once with cold 1X PBS.
2. Suspend the pellet with 500 μl of cold 5% (w/v) Metaphosphoric acid per 2-5 x 106 cells. Mix thoroughly by repeated pipetting. Homogenize or sonicate the cell suspension and store on ice for 5 minutes.
3. Transfer the suspension to a 1.5 ml tube and centrifuge at 12,000-14,000 x g for 5 minutes at 4˚C. Place the supernatant into a clean 1.5 ml tube. Store on ice if you intend to immediately assay for Glutathione, or freeze at -80˚C for future use.


B. Tissue Lysate Preparation
1. Remove as much blood as possible by perfusing the tissue with cold isotonic saline (150 mM NaCl) or 1X PBS containing heparin (0.16 mg/ml) to prevent coagulation.
2. Wash the tissue with cold isotonic saline (150 mM NaCl) or 1X PBS. Blot tissue on filter paper and weigh.
3. Add ice-cold 5% (w/v) Metaphosphoric acid (20 ml/g tissue) and homogenize using a cold glass or teflon pestle.
4. Centrifuge the homogenate at 12,000-14,000 x g for 10-15 minutes at 4˚C.
5. Collect the clarified supernatant. Store on ice if you intend to immediately assay for Glutathione, or freeze at -80˚C for future use.


C. Erythrocyte Lysate Preparation
1. Collect blood in Vacutainers containing heparin or sodium citrate as anticoagulant. Centrifuge at 3,000 x g for 10-15 minutes at 4˚C.
2. Discard as much of the plasma supernatant as possible. Remove the white buffy coat (leukocytes) on the surface of the erythrocytes.
3. Resuspend the erythrocyte pellet in four volumes of ice-cold 5% (w/v) Metaphosphoric acid. Mix thoroughly and store on ice for 15 minutes.
4. Centrifuge the suspension at 12,000-14,000 x g for 10-15 minutes at 4˚C.
5. Collect the clarified supernatant. Store on ice if you intend to immediately assay for Glutathione, or freeze at -80˚C for future use.


D. Whole Blood Lysate Preparation
1. Collect blood in tubes containing heparin or sodium citrate as anticoagulant.
2. Add four volumes of ice-cold 5% (w/v) Metaphosphoric acid. Mix thoroughly and store on ice for 15 minutes.
3. Centrifuge at 12,000-14,000 x g for 10-15 minutes at 4˚C.
4. Collect the clarified supernatant. Store on ice if you intend to immediately assay for Glutathione, or freeze at -80˚C for future use.

E. Urine, Plasma, and Saliva Lysate Preparation
1. Collect urine, plasma or saliva and immediately add four volumes of ice-cold 5% (w/v) Metaphosphoric acid. Mix thoroughly and store on ice for 15 minutes.
2. Centrifuge at 12,000-14,000 x g for 10-15 minutes at 4˚C.
3. Collect the clarified supernatant. Store on ice if you intend to immediately assay for Glutathione, or freeze at -80˚C for future use.


VIII. Assay Protocol


A. Total Glutathione Assay
1. Immediately prior to assay, dilute each experimental sample 10-fold with 1X Assay Buffer. Some biological specimens such as whole blood, liver, or red blood cells may need to be diluted 20-fold, 40-fold, or more.
2. Set up the Glutathione standard curve:
A. Add 50 μl of 1X Assay Buffer to all the wells in rows A through E, columns 1, 2, and 3 of the microtiter plate (see Figure 2).
B. Add 50 μl of the 4 μM GSSG to wells A1, A2, and A3 with a multichannel pipetor. Mix well by pipeting the solution up and down at least ten times.
C. Transfer 50 μl from wells A1, A2 and A3 to wells B1, B2, and B3, respectively. Mix well at least 10 times and transfer 50 μl from row B to row C. Continue in this fashion to row D. Mix and discard the last 50 μl from row D. Wells E1, E2, and E3 are set aside as blank wells. The GSSG content in rows A, B, C, and D, is 100 pmoles/well, 50 pmoles/well, 25 pmoles/well, and 12.5 pmoles/well, respectively.
3. Add 50 μl of your diluted experimental samples to the wells in columns 4 to 12.
Note: It may be necessary to make serial dilutions of your extracts to obtain a satisfactory change in absorbance readings with time. A sample dilution scheme is shown in Figure 2.
4. Prior to the next step, set up the parameters of your plate reader to measure absorbance at 405 nm or 414 nm and to read the required wells.
5. Using a multichannel pipetor, add 150 μl of freshly-prepared Reaction Mix to each well (see Section VI.2).
6. Immediately record the absorbance in the wells at 405 nm or 414 nm using a plate reader at 1 minute intervals over a 10 minute period.
Note: If you intend to use all the wells on one plate in the assay, it may be necessary to record the absorbance at 2 minute intervals.

B. Oxidized Glutathione Assay
1. Add 1 μl of 2M 4-vinylpyridine per 50 μl of sample and 4 μM GSSG. Incubate for one hour at room temperature (cell lysates should be diluted at least 1:10 prior to 4-vinylpyridine treatment).
2. Serially dilute the 4-vinylpyridine-treated GSSG standard as described above in the total glutathione assay protocol (Step VIII. A.2).
3. Serially dilute your 4-vinylpyridine-treated experimental samples as described above in the total glutathione assay protocol (Step VIII. A.3).
4. Follow steps 4, 5, and 6 as described above for the total glutathione assay.


IX. Data Interpretation


A. Determination of Total Glutathione Concentration
1. Take the average of the triplicate absorbance readings for each standard, sample, and blank at each time point.
2. Plot the average of each standard, sample, and background absorbance (A405 nm) versus incubation time and determine the slope from the linear portion of each curve (Figure 3).
3. Subtract the background slope from the slopes of the standards and the experimental samples.
4. Plot the net slopes of the GSSG standards versus pmoles of Glutathione (Figure 4).
5. Compare the net slopes of the experimental samples with those of the standard curve from Figure 4 to determine the pmoles of GSSG (equivalent to total glutathione) for each experimental sample.


B. Determination of Oxidized Glutathione Concentration

 

 

1. Follow the procedure described above for generating the Standard GSSG curves (Figures 3 and 4) for the 4-vinylpyridine treated standards.
2. Compare the net slopes of the 4-vinylpyridine-treated experimental samples with those of the 4-vinylpyridine-treated standards curve from Figure 4 to determine the pmoles of oxidized Glutathione for each experimental sample.
3. Subtract the pmole of oxidized glutathione in your sample from the pmole of total glutathione to obtain the pmole of reduced glutathione in your sample. Reduced GSH = Total glutathione - oxidized GSSG

X. References


1. Onyango IG, and Khan SM. Oxidative stress, mitochondrial dysfunction, and stress signaling in Alzheimer's disease. Curr Alzheimer Res. 2006 3:339-349
2. Medina S, Martinez M, Hernanz A. 2003. Antioxidants inhibit the human cortical neuron apoptosis induced by hydrogen peroxide, tumor necrosis factor alpha, dopamine and beta-amyloid peptide 1-42. Free Radic Res. 36:1179-84.
3. Julius M, Lang CA, Gleiberman L, Harburg E, DiFranceisco W, Schork A. 1994. Glutathione and morbidity in a community-based sample of elderly., J Clin epidemiol. 47: 1021-1026.
4. Mytilineou C, Kramer BC, Yabut JA. 2002. Glutathione depletion and oxidative stress. Parkinsonism Relat Disord. 8:385-387.


XI. Troubleshooting


XII. Related Products


Catalog # Description Size
4870-500-6 10X PBS, pH 7.4 6 X 500 ml
7500-100-K Superoxide Dismutase Assay Kit 100 tests
7501-500-K HT Superoxide Dismutase Assay Kit 480 tests
7510-100-K Glutathione Reductase Assay Kit 100 tests
7513-500-K HT Glutathione Reductase Assay Kit 480 tests
7512-100-K HT Glutathione Peroxidase Assay Kit 480 tests

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