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48-602MAG
Buffer Detection Kit for Magnetic Beads
1 Kit
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Description
Overview
A spectrophotometric assay kit that is specific for reduced glutathione (GSH) or total mercaptans. Absorbance is read at ~400 nm for GSH and ~356 nm for total mercaptens.
Note: 1 T = 1 test.
Catalogue Number
354102
Brand Family
Calbiochem®
Materials Required but Not Delivered
• Glutathione, Reduced (GSH) (Cat. No. 3541), purity >98% • Metaphosphoric acid (Cannot be substituted by any other acid), purity 33-37% • Spectrophotometer with absorbance measurable at 356 and 400 nm (from 0 to 2 absorbance unit). • Spectrophotometric cuvettes with 1-ml volume and 1-cm path length. • Cuvettes made of glass or plastic are suitable.
References
References
Ji, L.L., and Fu, R. 1992. J. Appl. Physiol.72, 549. Anderson M.E. 1989. Enzymatic and chemical methods for the determination of Glutathione; In: Glutathione: chemical, biochemical and medical aspects, Vol. A, Dolphin D., Poulson R. and Avramovic O. Eds., John Wiley and Sons, pp. 339-365. Dolphin D., et al. 1989. Glutathione: Chemical, Biochemical and Medical Aspects, Vols A & B, J. Wiley and Sons.
Product Information
Detection method
Colorimetric
Form
100 Tests
Format
Cuvette
Kit contains
A Chromogen, 30% NaOH, Buffer, and a user protocol.
Do not breathe fumes. In case of contact with eyes, rinse immediately with plenty of water and seek medical advice. Take off immediately all contaminated clothing. Wear suitable protective clothing, gloves and eye/face protection.
Product Usage Statements
Intended use
The Calbiochem® Glutathione Assay Kit makes it possible to assay for glutathione with only one sampling and one colorimetric measurement. A modification of this method makes it possible to assay other mercaptans. This alternate protocol is based on the measurement of substitution products that absorb maximally at 356 nm in the absence of reagent R2. Due to its ease of use, this method is well adapted for the quantification of glutathione and total mercaptans in a large number of biological samples. The main advantage of the method is that it does not require any enzyme as a reagent.
Storage and Shipping Information
Ship Code
Blue Ice Only
Toxicity
Multiple Toxicity Values, refer to MSDS
Hazardous Materials Attention:
Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.
Storage
+2°C to +8°C
Storage Conditions
Upon arrival store the entire kit contents at 4°C. • All solutions of reagents and buffer have been tightly sealed and are stable if properly stored between 4°C. Do Not Freeze. Protect from light. • After removing the required amount of each reagent for immediate use, all bottles should be tightly closed and stored between 2-4°C. Do not leave the reagent bottles open or at room temperature.
Protect from Light
Protect from light
Do not freeze
Yes
Packaging Information
Transport Information
Supplemental Information
Kit contains
A Chromogen, 30% NaOH, Buffer, and a user protocol.
Specifications
Global Trade Item Number
Catalogue Number
GTIN
354102-100T
04055977193275
Documentation
Glutathione Assay Kit Certificates of Analysis
Title
Lot Number
354102
References
Reference overview
Ji, L.L., and Fu, R. 1992. J. Appl. Physiol.72, 549. Anderson M.E. 1989. Enzymatic and chemical methods for the determination of Glutathione; In: Glutathione: chemical, biochemical and medical aspects, Vol. A, Dolphin D., Poulson R. and Avramovic O. Eds., John Wiley and Sons, pp. 339-365. Dolphin D., et al. 1989. Glutathione: Chemical, Biochemical and Medical Aspects, Vols A & B, J. Wiley and Sons.
Upon arrival store the entire kit contents at 4°C. • All solutions of reagents and buffer have been tightly sealed and are stable if properly stored between 4°C. Do Not Freeze. Protect from light. • After removing the required amount of each reagent for immediate use, all bottles should be tightly closed and stored between 2-4°C. Do not leave the reagent bottles open or at room temperature.
Intended use
The Calbiochem® Glutathione Assay Kit makes it possible to assay for glutathione with only one sampling and one colorimetric measurement. A modification of this method makes it possible to assay other mercaptans. This alternate protocol is based on the measurement of substitution products that absorb maximally at 356 nm in the absence of reagent R2. Due to its ease of use, this method is well adapted for the quantification of glutathione and total mercaptans in a large number of biological samples. The main advantage of the method is that it does not require any enzyme as a reagent.
Background
Oxidative stress occurs in most, if not all, human diseases. Our understanding of the role played by reactive oxygen species in the progression of pathological changes is still evolving. Convenient, accurate and reproducible methodologies to measure oxidative stress parameters have been lacking.
Glutathione (γ-glutamylcysteinylglycine or GSH) is a naturally occuring tripeptide whose nucleophilic and reducing properties play a central role in metabolic pathways, as well as in the antioxidant system of most aerobic cells. GSH plays a critical role as a coenzyme with a variety of enzymes including, glutathione peroxidase, glutathione S-transferase, and thiol transferase. GSH also plays major roles in drug metabolism, calcium metabolism, the γ-glutamyl cycle, blood platelet, and membrane functions. In addition, GSH is crucial to a variety of life processes, including the detoxification of xenobiotics, maintenance of the -SH level of proteins, thiol-disulfide exchange, removal of hydroperoxides and free radicals, and amino acid transport across membranes. Physiological values of intracellular GSH generally range from 1 to 10 mM. Although many methods have been described for the assay of GSH, the reliable ones are labor intensive and not easy to use.
Principles of the assay
The Calbiochem® Glutathione Assay Kit is based on a chemical reaction which proceeds in two steps. The first step leads to the formation of substitution products (thioethers) between a patented reagent, R1 (4-chloro-1-methyl-7-trifluromethyl-quinolinium methylsulfate), and all mercaptans (RSH) which are present in the sample:
Figure 1: RSH Sample
The second step is a β-elimination reaction that takes place under alkaline conditions. This reaction is mediated by reagent R2 (30% NaOH) which specifically transforms the substitution product obtained with GSH into a chromophoric thione with a maximal absorbance at 400 nm. This assay kit method is easier to use than the monobromobimane method and is more specific than the DTNB method, which picks up all thiols as well as some reactive amino acid species.
Figure 2: Sample 2
Materials provided
• Reagent (R1) (Kit Component No. KP0601): Solution of chromogenic reagent in HCl; 1 x 5.5 ml • Reagent (R2) (Kit Component No. KP0602): 30% NaOH. 1 X 10 ml • Buffer (Solution 3) (Kit Component No. KP0603): Potassium phosphate containing diethylenetriamine pentaacetic acid (DTPA) and lubrol. 1 X 100 ml Note: These three solutions are ready for use.
Materials Required but not provided
• Glutathione, Reduced (GSH) (Cat. No. 3541), purity >98% • Metaphosphoric acid (Cannot be substituted by any other acid), purity 33-37% • Spectrophotometer with absorbance measurable at 356 and 400 nm (from 0 to 2 absorbance unit). • Spectrophotometric cuvettes with 1-ml volume and 1-cm path length. • Cuvettes made of glass or plastic are suitable.
Precautions and recommendations
• Do not smoke, eat or drink in areas where reagents and samples are manipulated. • Wear disposable gloves when handling reagents and samples. • Do not pipette by mouth. • In case of accidental exposure of skin, mucous membranes, or eyes, to reagents R1 or R2, thoroughly wash the exposed area with water for 15 min and consult a physician. • Reagent R2 contains 30% sodium hydroxide and can cause severe burns.
Preparation
Erythrocyte Lysate
1. Centrifuge a minimum of 500 µl of whole blood at 2500 X g at 4°C for 5 min. 2. Discard plasma supernatant. If not assayed immediately, store erythrocyte pellet at -70°C (erythrocyte pellet can be stored at -70°C for 15 days). 3. Resuspend erythrocyte pellet in 4 volumes of MPA working solution, 0-4°C. 4. Thoroughly mix and centrifuge at 3000g at 4°C for 10 min. 5. Collect the upper clear aqueous layer and keep at 0-4°C for the assay (within 1 h).
Figure 3: Figure 4
Liver Homogenates
1. Wash tissue in 0.9% NaCl solution. 2. Blot tissue on paper and weigh. 3. Mince tissue in ice-cold MPA working solution. 4. Homogenize minced tissue. 5. Centrifuge homogenate at 3000 x g, 4°C for 10 min. 6. Collect the upper clear aqueous layer* and keep at 0-4°C for the assay (within 1 h). *Cloudy supernatant should be filtered through 0.2 µm filters.
Hepatocyte Lysates
1. Resuspend hepatocyte* pellet, from rats or mice, in 500 µl of ice-cold MPA working solution. 2. Homogenize cell suspension. 3. Centrifuge homogenate at 3000 x g, 4°C for 10 min. 4. Collect the upper clear aqeous layer and keep at 0-4°C for the assay (within 1 h). *Approximately 2.5-3.5 x 106 cells are used (5-8 mg of total protein).
Detailed protocol
Before each series of measurements: • Adjust the spectrophotometer absorbance to zero at 400 nm, with buffer only (solution 3). • Perform three independent measurements of blank absorbance (A) at 400 nm. The mean value of A will be subtracted from the absorbance values obtained with sample (A). The
blanks should be measured once only after 10 min of incubation.
For each measurement, the reaction mixture is prepared as follows: 1. Take an initial volume (Vi) sample (20-300 µl). 2. Bring to 900 µl final volume with buffer (Volume of solution 3 = 900 µl-Vi). 3. Add 50 µl of solution R1 and thoroughly mix. 4. Add 50 µl of solution R2 and thoroughly mix. 5. Incubate at 25 ± 3°C for 10 min in the dark*. 6. Measure the final absorbance (A) at 400 nm. .*Samples are stable for 1 h after the 10-min-incubation if they are kept in the dark.
Before each new series of assays, prepare a standard curve with at least five distinct concentrations of GSH. These five concentrations should cover the range 20-100 µmol/l in the reaction medium (spectrophotometric cuvette). Prepare a MPA working solution by dissolving 5 grams MPA in 100 ml water. Prepare a 0.5 mmol/l GSH working standard solution by dissolving GSH into MPA working solution.
Figure 4: Figure 6
Table 1: GSH Standard Curve
A least-squares linear regression should demonstrate that the absorbance at 400 nm (A) is a linear function of GSH concentration. The apparent molar extinction coefficient, ∑, of the measured product is equal to the slope of the corresponding straight line. An example of a standard curve obtained at 400 nm.
Figure 5: Example of standard curve obtained at 400 nm at 25°C.
Calculations
Measurement of Total Mercaptans (RSH): Assay at 356 nm
The Calbiochem Glutathione Assay Kit can be used for the measurement of other mercaptans (RSH), which include GSH. The assay is carried out in the absence of reagent R2 at 356 nm. The absorbance at 356 nm is a linear function of [RSH] concentration in the sample, but it is not GSH-specific. If the sample essentially contains GSH and a single other mercaptan i.e., N-acetylcysteine, these two mercaptans can be assayed by using the same single sample for two measurements. The first measurement is made at 356 nm before the addition of reagent R2 as described below, and the second measurement is made, after the addition of R2, at 400 nm.
Adjust the spectrophotometer absorbance to zero at 356 nm with buffer only (solution 3). The reaction mixture is prepared as in Section 3, with the omission of step 4. The absorbance (A) is measured at 356 nm.
A standard curve must be prepared with the corresponding mercaptan in Table 2, to calculate the concentration. Figure 2 gives two examples of standard curves obtained with glutathione and Nacetylcysteine at 356 nm.
Figure 6: Example of standard curve obtained at 356 nm at 25°C.
GSH Concentration The calculation is based on the following equation: [GSH] = {(A-A0)/(E x l)} x D where: [GSH] is the initial glutathione concentration in the sample, expressed as molar concentration.
A and A0 are the absorbances measured in the presence and in the absence of sample, respectively.
E is the apparent molar extinction coefficient of the product measured at 400 nm.
l is the optical path (cm).
D is the dilution factor of the sample.
Total Mercaptan Concentration The calculation is based on the modified version of the equation used for GSH: [total RSH] = { (A - A0) / (E x I) } x D where : [total RSH] is the concentration of total mercaptan in the sample.
A and A0 are the absorbances measured in the presence and in the absence of sample, respectively.
E is the apparent molar extinction coefficient of the product measured at 356 nm. I is the optical path (cm).
D is the dilution factor of the sample.
Note: • Do not add reagents R1 and R2 in reverse order.
• The temperature, (25 ± 3°C), should be kept constant throughout the experiment. • The final reaction volume (1 ml) should not vary from one measurement to another. • The absorbance at 400 nm is proportional to glutathione concentration. It is stable for min, provided that the reaction mixture is kept in the dark. • The sensitivity of the assay for mercaptans at 356 nm is of the same order of magnitude • as that of glutathione at 400 nm as shown in Table 2.
Sensitivity
5.0 µM RSH
Sensitivity Notes
From 30 repeated measurements performed on the control ([RSH] = 0) on the same day, under the same experimental conditions, the detection limit of the assay was 5 µmol/l in the final reaction mixture (spectrophotometric cuvette). For example, using the maximum volume of 300 µl of a sample would therefore give a detection limit for glutathione of about 17 µmol/l.
Precision
With a series of 30 measurements performed on the same day and under the same experimental conditions, using GSH (20-200 µM), the standard error of the mean value (SEM) was less than 2%.
Reproducibility
With the same experiments performed twice at three-day intervals, using the same samples, the new SEM calculated on the two measurement series was lower than 2%.
Registered Trademarks
Calbiochem® is a registered trademark of EMD Chemicals, Inc. Interactive Pathways™ is a trademark of EMD Chemicals, Inc.
