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| Catalog # | Availability | Size | Quantity | Unit Price | Save For Later Wish List | |
|---|---|---|---|---|---|---|
| EKFT19250UV-96 | 2 weeks | 96T | $600.00 |
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| EKFT19250UV-48 | 2 weeks | 48T | $420.00 |
Product Overview | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Name | Reduced Glutathione (GSH) colorimetric Assay Kit | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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| Accession (Primary) | CAS: 70-18-8 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Reactivity | Universal | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Sensitivity | 1.20 umol/L | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Assay Range | 1.56-100 umol/L | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Detection Method | Colorimetric Assay | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Sample type(s) | Serum, Plasma, Cell Culture Supernatant, cell or tissue lysate, Other liquid samples | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Product Features | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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| Principle of the Assay | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| The spectrophotometric/microplate reader assay method for glutathione (GSH) involves oxidation of GSH by the 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) to form the yellow derivative 5′-thio-2-nitrobenzoic acid (TNB), measurable at 412 nm. The lowest detection for GSH is 1.2umol in a 96-well plate. The concentration of GSH in the samples is determined by comparing the OD of the samples to the standard curve. The concentration of GSH is proportional to the OD value. The kit is rapid and the whole procedure takes no longer than 20 min including reagent preparation. It can assay GSH in whole blood, plasma, serum, lung lavage fluid, cerebrospinal fluid, urine, tissues and cell. Reaction mechanism： [Picture_0] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Kit Components and Storage | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
The sealed kit can be stored at 2-8 ℃. The storage condition for opened kit is specified in the table below:
Note: The liquid reagent bottle contains slightly more reagent than indicated on the label. Please use pipette accurately measure and do proportional dilution.Required Instruments and Reagents 1. Microplate reader (wavelength: 405 or 414nm) 2. Precision single (0.5-10μL, 5-50μL, 20-200μL, 200-1000μL) and multi-channel pipette with disposable tips(calibration is required before use.) 3. Sterile tubes and Eppendorf tubes with disposable tips 4. 10mM PBS PH7.2-8.0 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Sample Collection and Storage | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 1. Serum Place whole blood sample at room temperature for 2 hours or at 2-8°C overnight. Centrifuge for 20min at 1000xg and collect the supernatant to detect immediately. Or you can aliquot the supernatant and store it at -20°C or -80°C for future’s assay. 2. Plasma EDTA-Na2/K2 is recommended as the anticoagulant. Centrifuge samples for 15 minutes at 1000×g 2-8°C within 30 minutes after collection. Collect the supernatant to detect immediately. Or you can aliquot the supernatant and store it at -20°C or -80°C for future’s assay. For other anticoagulant types and uses, please refer to the sample preparation guideline. 3. Tissue Sample Tissue samples are required to be made into homogenization. Protocol is as below: 3.1. Place the target tissue on the ice. Remove residual blood by washing tissue with pre-cooling PBS buffer (0.01M, pH=7.4). Then weigh for usage. 3.2. Use lysate to grind tissue homogenates on the ice. The adding volume of lysate depends on the weight of the tissue. Usually, 9mL PBS would be appropriate to 1 gram tissue pieces. 3.3. Do further process using ultrasonic disruption or freeze-thaw cycles (Ice bath for cooling is required during ultrasonic disruption; Freeze-thaw cycles can be repeated twice.) to get the homogenates. 3.4. Homogenates are then centrifuged for 5 minutes at 5000×g. Collect the supernatant to detect immediately. Or you can aliquot the supernatant and store it at -20°C or -80°C for future’s assay. 3.5. Determine total protein concentration by BCA kit for further data analysis. Usually, total protein concentration for assay should be within 1-3mg/ml. Notes: PBS buffer or the RIPA lysis can be used as lysates. Avoid using any reagents containing DTT Or reducing agent due to their severe interfere with kits’ working. 4. Cell Culture Supernatant Collect the supernatant: Centrifuge at 2500 rpm at 2-8℃ for 5 minutes, then collect clarified cell culture supernatant to detect immediately. Or you can aliquot the supernatant and store it at -80°C for future’s assay. Cell Lysate Suspension Cell Lysate: Centrifuge at 2500 rpm at 2-8℃ for 5 minutes and collect cells. Then add pre-cooling PBS into collected cell and mix gently. Recollect cell by repeating centrifugation. Add 0.5-1ml cell lysate. Lyse the cell on ice for 30min-1h or disrupt the cell by ultrasonic disruption. 5.2. Adherent Cell Lysate: Absorb supernatant and add pre-cooling PBS to wash three times. Add 0.5-1ml cell lysate. Scrape the adherent cell with cell scraper. Lyse the cell suspension added in the centrifuge tube on ice for 30min-1h or disrupt the cell by ultrasonic disruption. 5.3. During lysate process, use the tip for pipetting or intermittently shake the centrifugal tube to completely lyse the protein. Mucilaginous product is DNA which can be disrupted by ultrasonic cell disruptor on ice. (3~5mm probe, 150-300W, 3~5 s/time, 30s intervals for 1~2s working). 5.4. At the end of lysate or ultrasonic disruption, centrifuge at 10000rpm at 2-8℃ for 10 minutes. Then, the supernatant is added into EP tube to detect immediately. Or you can aliquot the supernatant and store it at -80°C for future’s assay. Notes: Read notes in tissue sample. 6. Other Biological Sample Centrifuge samples for 15 minutes at 1000×g at 2-8℃. Collect the supernatant to detect immediately. Or you can aliquot the supernatant and store it at -80°C for future’s assay. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Notes for Samples | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Blood collection tubes should be disposable and non-endotoxin. Avoid to use hemolyzed and lipemia samples. The best sample storage condition: less than 5 days at 2-8℃; within 6 months at -20℃; within 2 years at -80℃. Stored in liquid nitrogen for a longer storage. When melting frozen samples, rapid water bath at 15-25℃ can decrease the effect of ice crystal (0℃) on the sample. After melting, centrifuge to remove the precipitate, and then mix well. The detection range of this kit is not equivalent to the concentration of analyze in the sample. For analyses with higher or lower concentration, please properly dilute or concentrate the sample. 4. Pretest is recommended for special samples without reference data to validate the validity. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Precautions for Kits | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 1. After opening the kit, please refer to the table of storage condition for GSH standards (Dampness may decrease the activity.). If any component is missing or damaged during the assay or storage, please contact us for ordering a new one to replace. (e.g. GSH standard) 2. Sterile and disposable tips are required during the assay. After use, the reagents bottle cap has to be tightened to avoid the microbial contamination and evaporation. 3. Before confirmation, reagents from other batches or sources should not be used in this kit. 4. Don't reuse tips and tubes to avoid cross contamination. 5. Please wear the lab coat, mask and gloves to protect yourself, during the assay. Especially, for the detection of blood or other body fluid sample, please follow regulations on safety protection of biological laboratory. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Recommended Sample Dilution Ratio | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Random individual test data:
If other dilution ratio for your sample model is required, please refer to the universal dilution ratio below. (The ratio is suitable for single-well assay. For duplicate assay, please follow the calculation: volume of sample and diluent x number of duplicate well) For 2 fold dilution (1/2): One step dilution. Add 60μL sample into 60μL PBS and mix gently. For 5 fold dilution (1/5): One step dilution. Add 24μL sample into 96μL PBS and mix gently. For 10 fold dilution (1/10): One step dilution. Add 12μL sample into 108μL PBS and mix gently. For 20 fold dilution (1/20): One step dilution. Add 6μL sample into 114μL PBS and mix gently. For 50 fold dilution (1/50): One step dilution. Add 3μL sample and 47μL PBS into 100 μL PBS and mix gently. For 100 fold dilution (1/100): One step dilution. Add 3μL sample and 177μL PBS into 120μL PBS and mix gently. For 1000 fold dilution (1/1000): Two step dilution. Create a 50-fold dilution first. Then, create a 20-fold dilution and mix gently. For 10000 fold dilution (1/10000): Two step dilution. Create a 100-fold dilution first. Then, create the same dilution again and mix gently. For 100000 fold dilution (1/100000): Three step dilution. Create a 50-fold dilution and 20-fold dilution respectively .Finally, create a 100-fold dilution and mix gently. Notes: The volume in each dilution is not less than 3μL. Dilution factor should be within 100 fold. Mixing during dilution is required to avoid foaming. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Reagent Preparation and Storage | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| The kit is ready for immediate use when taken out of the refrigerator. 10mM GSH standard storage solution Centrifuge GSH standards tube for 1min at 10000xg. Label it as 10mM GSH. Add 1ml Standard stock solution buffer into the standard tube. Tighten the tube cap and invert the tube several times to mix gently. (Or you can mix it using a low speed vortex mixer for 20 seconds.) Centrifuge the tubes for 10s at 1000xg, making the liquid towards the bottom of tube. 2. Standards 2.1. Label an EP tube with zero tube. Add 990ul Assay Dilution Buffer and 10ul 10mM GSH standard storage solution into it. Invert the tube several times to mix gently. (Or you can mix it using a low speed vortex mixer for 3-5 seconds.) 2.2. Centrifuge the tubes for 1min at 1000xg, making the liquid towards the bottom of tube and removing possible bubbles. 2.3. Standard dilution: Label 7 EP tubes with 1/2, 1/4, 1/8, 1/16, 1/32, 1/64 and blank respectively. Add 0.3ml of the Assay Dilution Buffer into each tube. Add 0.3ml solution from zero tube into 1/2 tube and mix them thoroughly. Transfer 0.3ml from 1/2 tube into 1/4 tube and mix them thoroughly. Transfer 0.3ml from 1/4 tube into 1/8 tube and mix them thoroughly, so on till 1/64 tube. Now blank tube only contain 0.3ml Assay Dilution Buffer. The standard concentration from zero tube to blank tube is 100umol/L, 50umol/L, 25umol/L, 12.5umol/L, 6.25umol/L, 3.125umol/L, 1.562umol/L, 0umol/L. [Picture_1] Notes: The 10mM GSH standard storage solution can be stored at -20 ° C for 6 months or at 2-8 ° C for 2 months. Other diluted working solutions containing standards should be used in 2h. 3. Preparation of DTNB Working Solution The working solution should be prepared within 5min before the assay and can't be stored for a long time. 3.1. Calculate required total volume of the working solution: 100ul/well x quantity of wells. (It's better to prepare additional 100ul-200ul.) 3.2. Centrifuge for 10s at 1000xg in low speed and bring down the concentrated DTNB (20X) to the bottom of tube. 3.3. Dilute the DTNB with Assay Dilution Buffer at 1/20 and mix them thoroughly. (e.g. Add 50ul concentrated DTNB into 950ul Assay Dilution Buffer.) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Detailed Assay Procedure | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| When diluting samples and reagents, they must be mixed completely. It's recommended to plot a standard curve for each test. 1. Set standard, pilot samples, control (blank) wells on the plate respectively, and then, records their positions. It's recommended to measure each standard and sample in duplicate to decrease experimental errors. 2. Standards and samples loading: Aliquot 100ul of zero tube, 1st tube, 2nd tube, 3rd tube, 4th tube into each standard well. Also add Assay Dilution Buffer into the control (blank) well. Then, add 100ul pilot samples into each sample well. 3. DTNB Working Solution loading: Add 100ul DTNB Working Solution into each well. Then gently shake the plate to mix the working solution and incubated for 3 minutes at room temperature. 4. OD Measurement: Read the O.D. absorbance at 405nm or 414nm in a microplate reader immediately. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Calculation of Results | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 1. Calculate the mean OD value of the duplicate readings for each standard, control, and sample. 2. Create a Linear Fit curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis. Alternatively, you can use the curve fitting software offered by the microplate reader (e.g. Thermo SkanIt RE software, Curve Expert 1.3 or 1.4). 3. Calculate the sample concentration by substituting OD value into the standard curve. Diluted samples should be multiplied by the relevant dilution ratio.
Note：f (sample dilution factor), C (total protein concentration of the samples) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Typical Data & Standard Curve | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| This product has been tested by Quality Control Department and meets performance specifications mentioned in the manual. (The humidity in the laboratory is 20%-60%, and the temperature is 18°C -25°C.) The following assay data are provided for reference, since experimental environment and operation are different. The establishment of standard curve depends on your own assay.
[Picture_2] | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Precision | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Intra-assay Precision: samples with low, medium and high concentration are tested 20 times on same plate. Inter-assay Precision: samples with low, medium and high concentration are tested 20 times on three different plates.
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| Recovery | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Add a certain amount of GSH into the sample. Calculate the recovery by comparing the measured value with the expected amount of GSH in the sample.
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| Linearity | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Dilute the sample with a certain amount of GSH at 1:2, 1:4 and 1:8 to get the recovery range.
Troubleshooting If the result is unsatisfied, please take a screenshot for the staining result and store the OD data. Keep used strips as well the rest reagents. Contact us to solve your problem with the kit’s catalogue number and batch number. You can also refer to the following table to check the reason.
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| Declaration | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Limited to current conditions and scientific techniques, all raw materials are not completely identified and analyzed. This product may have a technology-related quality risk. During the Elisa kit development, some endogenous interferons(not all) in the biological sample have been removed or decreased. The final assay result is related to the validity of reagents, experimental operation and environment. Our company is only responsible for this kit, excluding sample consumption during using this kit. Before use, please consider and prepare enough samples required by the assay. To get a satisfied assay result, please use all reagents offered by this kit. Don't use any product from other vendors. Strictly follow instructions of this manual. During assay procedure, incorrect reagents preparation and parameter setting of the microplate reader may result in the abnormal result. Before assay, please read this manual carefully and adjust instruments. Even if the assay is performed by the same person, results in two independent assays may be different. Thus, each step in the assay should be controlled to ensure the reproducibility. Before delivery, this kit is subject to the strict QC. Influenced by transportation conditions and experimental devices, the assay result got by the customer may be different from original data. Inter-assay CV between different batches may be caused by reasons before. This kit is not compared to similar kits from other vendors or methods for testing the same detection target. Thus, assay results may be inconsistent. This kit allows for research use only. For IVD or other purposes, our company is not responsible for relevant consequences and doesn't bear any legal liability. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Target Information: ( CAS: 70-18-8 ) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Background |
CAS: 70-18-8 |
Thousands of laboratories across the world have published research that depended on the performance of antibodies from ADMEbio to advance their research.
Check out links to articles that cite our products in major peer-reviewed journals, organized by research category.
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ADMEbio Team
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ADMEbio Team
