| Description | Glutathione is a tripeptide containing a γ-amide bond and a sulfhydryl group, composed of glutamate, cysteine, and glycine. It is widely found in animal and plant tissues and microorganisms. In living organisms, it helps maintain normal immune system function and has antioxidant and Glutathione is a tripeptide containing a γ-amide bond and a sulfhydryl group, composed of glutamate, cysteine, and glycine. It is widely found in animal and plant tissues and microorganisms. In living organisms, it helps maintain normal immune system function and has antioxidant and detoxifying effects. Glutathione exists in two forms: reduced (GSH) and oxidized (GSSG). GSSG, also known as glutathione disulfide, is formed by the oxidation of two glutathione molecules. GSSG can be reduced back to GSH by glutathione reductase; therefore, it exists primarily in the reduced form in organisms. The ratio of reduced to oxidized glutathione (GSH/GSSG) serves as a key dynamic indicator for assessing the cellular redox state. Detection Principle: Endogenous GSH in the sample is masked by 2-vinylpyridine. Under the catalysis of glutathione reductase (GR), GSSG is reduced to GSH. The generated GSH then reacts with 5,5'-Dithiobis-(2-nitrobenzoic acid) (DTNB) to produce yellow-colored 5-thio-2-nitrobenzoic acid (TNB), which has a characteristic absorption peak at 412 nm. The GSSG content is quantified by measuring the change in absorbance. Detection Range: 1-20 µM Sensitivity: 1 µM Applicable Samples: Animal/plant tissues, blood cells, cells, bacteria, serum (plasma).O1492795Component96TStorageO1492795AExtraction Buffer70 mL×22-8℃O1492795BInhibitor210 µL-20℃. Store in the dark.O1492795CAssay Buffer20 mL2-8℃O1492795DGR14 µL2-8℃. Store in the dark.O1492795EGR Cofactor2 EA-20℃. Store in the dark.O1492795FChromogen2 EA2-8℃. Store in the dark.O1492795GStandard1 EA2-8℃. Store in the dark.User-Provided Instruments and ReagentsTypeNameNotesInstrumentMicroplate ReaderCapable of measuring absorbance at 412 nm.Consumables96-well MicroplateStandard transparent plate.ReagentsPBS / Deionized WaterFor washing samples / Preparing reagents.OthersHomogenizer (for tissue samples), water bath, ice bucket, low-temperature centrifuge, adjustable pipettes and tipsUsing a multichannel pipette for large-scale detection can improve efficiency.Experimental Procedure1. Reagent PreparationReagent NameReagent PreparationPrecautionsExtraction BufferReady-to-use; equilibrate to room temperature before use.Store at 4°C.Diluted Extraction BufferAdd 500 µL Extraction Buffer to 4.5 mL deionized water.Obtained by 10-fold dilution of Extraction Buffer.InhibitorReady-to-use; equilibrate to room temperature before use.Store at -20°C protected from light. Toxic and irritant; recommended to handle in a fume hood.Assay BufferReady-to-use; equilibrate to room temperature before use.Store at 4°C.GR DilutionBefore use, prepare by adding 1 µL GR to 20 µL deionized water per sample.Prepare freshly before use.GR Cofactor DilutionBefore use, add 1.5 mL deionized water to each vial; equilibrate to room temperature protected from light.After dissolution, store at -20°C protected from light for up to 1 month.Chromogen DilutionBefore use, add 1.5 mL deionized water to each vial; equilibrate to room temperature protected from light.After dissolution, store at 4°C protected from light for up to 1 month.GSSG StandardDissolve in 1 mL of Diluted Extraction Buffer.20 mM; After dissolution, aliquot and store at -20°C protected from light for up to 1 month.2. Standard PreparationTake 100 µL of the 20 mM GSSG standard and dilute with 900 µL Diluted Extraction Buffer to obtain a 2 mM GSSG standard solution.Take 10 µL of the 2 mM GSSG standard and dilute with 990 µL Diluted Extraction Buffer to obtain a 20 µM GSSG standard solution.Further dilute the standard as shown in the table below. A standard curve must be prepared for each experiment. Diluted standard solutions are unstable and must be used within 4 hours.Standard Working Solution20µM Standard (µL)Diluted Extraction Buffer (µL)Concentration (µM)110002028020163604012440608520804610902759513. Sample PreparationNote: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 10 days. Because the Extraction Buffer contains a protein precipitant, the supernatant cannot be used for protein concentration determination. If protein content needs to be measured, prepare another identical sample using deionized water instead of Extraction Buffer.3.1 Animal/Plant Tissue Samples:Use fresh tissue samples whenever possible. Weigh 0.1 g of tissue, add 1 mL of pre-cooled Extraction Buffer, and homogenize quickly on ice (pre-cool the homogenizer on ice). Centrifuge the homogenate at 8000 g, 4°C for 10 min. Collect the supernatant and keep on ice for detection.3.2 Serum/Plasma Samples:Use fresh serum (plasma) whenever possible. Centrifuge the collected serum (plasma) at 600 g, 4°C for 10 min. Within 30 minutes, aspirate the supernatant into another tube. Add an equal volume of Extraction Buffer, mix, then centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep on ice for detection.3.3 Cell or Bacterial Samples:Use fresh cells (bacteria) whenever possible; avoid using frozen cells (bacteria). Collect 5×10⁶ cells (bacteria). Wash twice with 1 mL of pre-cooled PBS (resuspend in PBS, centrifuge at 600 g, 4°C for 10 min). Add 3 times the volume of Extraction Buffer relative to the cell (bacterial) pellet to resuspend the cells (bacteria). Disrupt by ultrasound on ice (power 20% or 200 W, ultrasonicate for 3 s, interval 7 s, repeat 30 times). Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep on ice for detection.Note: Cells can also be extracted using a freeze-thaw method (not suitable for bacteria): Resuspend cells and subject to 2-3 rapid freeze-thaw cycles (freeze in liquid nitrogen, thaw in a 37°C water bath). Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep on ice for detection.4. Assay Steps4.1 Microplate Reader Preparation: Preheat for at least 30 minutes, set wavelength to 412 nm.4.2 Assay System Setup (Step 1 - Pre-treatment): Perform the following operations in 1.5 mL EP tubes. This step must be done in EP tubes. Do not add Inhibitor directly to the 96-well plate as it may corrode the plate. Inhibitor is toxic and irritant; recommended to handle in a fume hood.ReagentBlank Tube (µL)Standard Tube (µL)Test Tube (µL)Sample003Deionized Water30027Standard0300Inhibitor1.51.51.54.3 Mix well and incubate at 37°C for 30 minutes. This becomes the "Mixture".4.4 Assay System Setup (Step 2 - Reaction): Perform the following operations in a 96-well plate.ReagentBlank Well (µL)Standard Well (µL)Test Well (µL)Mixture212121Assay Buffer140140140GR Dilution222GR Cofactor Dilution202020Chromogen Dilution2020204.5 Absorbance Measurement: Mix thoroughly after addition. Read the absorbance at 412 nm (A1), recorded as A1 blank, A1 standard, and A1 test. Then incubate at 37°C protected from light for 10 minutes. Quickly read the absorbance at 412 nm again (A2), recorded as A2 blank, A2 standard, and A2 test. 5. Result Calculation The following provides both the derived formula and the simplified calculation formula, which are completely equivalent. 5.1 Data Processing Calculate ΔA = A2 - A1 for each. Then calculate ΔΔA standard = ΔA standard - ΔA blank And ΔΔA test = ΔA test - ΔA blank 5.2 Standard Curve Plotting 5.2 Standard Curve Plotting Plot the standard curve with standard concentration as the y-axis and ΔΔA standard as the x-axis. Substitute ΔΔA test into the equation to obtain the y value (µM). 5.3 Sample GSSG Content Calculation (1) Based on sample mass: GSSG (nmol/g) = y × V standard ÷ V sample × V extract ÷ W × n = 10 × y ÷ W × n (2) Based on cell or bacterial count: GSSG (nmol/10⁴) = y × V standard ÷ V sample × V extract ÷ 500 × n = 0.02 × y × V extract × n (3) Based on liquid volume: GSSG (nmol/mL) = y × V standard ÷ V sample × 2 × n = 20 × y × n (4) Based on protein concentration: GSSG (nmol/mg prot) = y × V standard ÷ V sample ÷ Cpr × n = 10 × y ÷ Cpr × n Parameter Description: 1 µM = 1 nmol/mL; V standard : Volume of standard added, 30 µL; V sample : Volume of sample added, 3 µL; V extract : Volume of Extraction Buffer added, 1 mL (for cells/bacteria, use the actual volume used); W: Sample mass, g; n: Sample dilution factor; Cpr: Sample protein concentration, mg/mL; 500: Cell or bacterial count, in units of 10⁴; 2: Dilution factor for liquid samples (added equal volume of Extraction Buffer).6. Result PresentationTypical Standard Curve: y = 8.0042x + 0.212, R² = 0.9997Example-1: 0.1 g of rat liver tissue was processed and assayed according to the procedure using a 96-well plate. Measured: ΔA test = A2 test - A1 test = 0.386 - 0.120 = 0.266 ΔA blank = A2 blank - A1 blank = 0.132 - 0.097 = 0.035 ΔΔA test = ΔA test - ΔA blank = 0.266 - 0.035 = 0.231 Substituting ΔΔA test into the standard curve equation gives y = 2.061 µM. Calculated based on sample mass: GSSG (nmol/g) = y × V standard ÷ V sample × V extract ÷ W × n = 10 × y ÷ W × n = 206.1 nmol/g.Precautions1. It is recommended to perform preliminary experiments using 2-3 samples expected to have significant differences before formal testing.2. The samples extracted with this kit are suitable for the detection of oxidized glutathione (GSSG). Because the extraction buffer contains a protein precipitant, the supernatant cannot be used for protein concentration determination. If protein content needs to be measured, prepare another identical sample using deionized water instead of the extraction buffer. For protein concentration determination, Aladdin BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) are recommended.3. This kit is compatible with spectrophotometer detection. Adjust the preparation volume of detection reagents proportionally according to the spectrophotometer's requirements.4. It is recommended to establish your own standard curve for improved accuracy. If not, you may refer to the typical standard curve formula provided in the results section for calculation.5. Biochemical reagents are generally irritating and biologically toxic. For your safety and health, please wear appropriate personal protective equipment (lab coat, mask, gloves, hair cap, etc.) throughout the experiment and perform experiments in a fume hood or biosafety cabinet.6. This product is for scientific research use only. Not intended for clinical diagnosis.Frequently Asked Questions Q: What should I do if the sample ΔA test is too high or too low? Frequently Asked Questions Q: What should I do if the sample ΔA test is too high or too low? A: If the sample ΔA test is greater than the ΔA standard of the 20 µM standard, the GSSG content in the sample is too high. Dilute the sample appropriately with deionized water (multiply by the dilution factor in the calculation). If the sample ΔA test is less than 0.005, increase the sample amount.Q: Can blood cell samples be detected?A: Yes, blood cell samples can be detected. Centrifuge the collected anticoagulated blood at 600 g, 4°C for 10 min. Discard the upper plasma and wash the pellet 2-3 times with 3 volumes of PBS (resuspend blood cells in PBS, centrifuge at 600 g, 4°C for 10 min). Add an equal volume of Extraction Buffer, mix, and let stand at 4°C for 10 min. Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep on ice for detection... Read More | DescriptionCAR10 is a kit that contains a selection of 10 carbohydrates/sugars: Arabinose, Fructose, Galactose, Glucose, α-Lactose, Maltose, Mannose, Ribose, Sucrose and Xylose, which may be used for general research, as reagents or as reference compounds in analytical procedures | D-Lactate, typically present in the bloodstream at nanomolar concentrations, is produced by an intestinal source or via the methylglyoxal pathway. In mammals, D-Lactate metabolism requires D-Lactate hydrogenase and is metabolized slowly, thus an increase in blood concentration levels can lead to D-Lactate, typically present in the bloodstream at nanomolar concentrations, is produced by an intestinal source or via the methylglyoxal pathway. In mammals, D-Lactate metabolism requires D-Lactate hydrogenase and is metabolized slowly, thus an increase in blood concentration levels can lead to acidemia and acidosis. The severity of this D-lactic acidosis can be associated with neurotoxic symptoms. Significant D-Lactate accumulations in the body can also be related to impaired metabolism and excretion.D-Lactate Colorimetric Assay kit has been used to determine the stereospecificity of lactate produced.Suitability: Suitable for use with samples of serum, plasma, cells, culture and fermentation media.Principle: In this assay, D-Lactate is specifically oxidized by D-Lactate hydrogenase and generates a proportional colorimetric product measured at 450 nm. The useful concentration range in samples is 0.1-10 mM D-Lactate... Read More | Apoptosis refers to the cell autonomous and orderly death controlled by genes to maintain the stability of the internal environment. Apoptosis is different from cell necrosis. Apoptosis generally refers to a programmed cell death process that occurs during the development of body cells or under the Apoptosis refers to the cell autonomous and orderly death controlled by genes to maintain the stability of the internal environment. Apoptosis is different from cell necrosis. Apoptosis generally refers to a programmed cell death process that occurs during the development of body cells or under the action of some factors through the regulation of intracellular genes and their products. Cell necrosis is a cell death process that is caused by strong physical and chemical or biological factors to cause disordered changes in cells. The difference between apoptosis and necrosis lies in the characteristic morphological and biochemical changes, including the changes of cell membrane permeability and nuclear chromatin, the contraction of cytoplasm and the loss of membrane asymmetry. The oxazole yellow/pi membrane permeability apoptosis detection kit produced by our company is a dual fluorescence detection kit based on oxazole yellow and PI dyes. This kit is suitable for fluorescence microscopy, flow cytometry, fluorescence microplate reader and other fluorescence detection systems. Oxazole yellow is a non cell membrane penetrating cyanine monomer green fluorescent dye with high affinity for DNA. It basically has no fluorescence when it is not bound to DNA, but can emit bright green fluorescence after binding to DNA. When apoptosis occurs, the permeability of cell membrane changes. At this time, oxazole yellow can enter the cell and bind to DNA, emitting bright green fluorescence. Therefore, it is often used for the detection of apoptosis. It should be noted that oxazole yellow can also stain dead cells, so it needs to be double stained with PI that specifically fluorescently stains dead cells to effectively determine apoptosis. PI (propidium iodide) is a red fluorescent dye that can stain DNA. It is an analog of pyridine bromide that releases red fluorescence after embedding double stranded DNA. Although PI cannot pass through the membrane of living cells, it can cross the damaged cell membrane of dead cells to stain nuclei. Therefore, oxazole yellow combined with PI can be directly used for the detection of apoptosis. Apoptotic cells show green fluorescence, dead cells show both red and green fluorescence positive, and living cells have little or no fluorescence.Components: Components O598364-50T A. Oxazole yellow dye 50 µL B. Propidium Iodide (PI) 50 µLUsage (using flow cytometry as an example):1. Cell preparation(1) For adherent cells, after trypsin digestion, resuspend in culture medium and wash once with pre cooled PBS; The digestion time of trypsin should not be too long to prevent false positives. Note: Digest with trypsin and allow the cells to recover in the optimal cell culture conditions and medium for about 30 minutes, then stain.(2) For suspended cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and wash once with pre cooled PBS.2. Cell stainingSuspend cells in pre cooled PBS, with a recommended cell count of 106 cells/mL per sample. Add 1 µ L Oxazole Yellow and 1 µ L to 1 mL of the samplePI， Gently blow and mix well. Incubate on ice in the dark for 30 minutes. Note: We suggest adding the following two experimental controls:Blank tube: negative control group cells, without dye, used to regulate voltage.Single staining tube: Positive control group cells were treated with only two tubes, Oxazole yellow and PI, for regulating compensation.3. Flow detectionAfter incubation, the sample can be directly detected by flow cytometry, or centrifuged at 1000 rpm for 5 minutes, the supernatant can be aspirated, and the sample can be resuspended in 1 mL of pre cooled PBS for flow cytometry detection. Oxazole yellow can be excited by a 488 nm laser, and the detected fluorescence emission spectrum is around 530 ± 30 nm (FITC channel), while the PI channel emission spectrum is around 617 nm (PI or PE channel).Product parameters：Oxazole yellow dye:ex/em = 491 / 509 nm (bound DNA); Propidium iodine:ex/em = 535 / 617 nm (combined with DMatters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. fluorescent dyes have quenching problems. Please try to avoid light to slow down fluorescence quenching. 3. for your safety and health, please wear experimental clothes and disposable gloves.Scope of application：Membrane permeability apoptosis assay... Read More | The Succinic Acid (Succinate) assay kit is suitable for the specific assay of succinic acid in wine, cheese, eggs, sauce and other food products. Succinic acid (or succinate) is found in all plant and animal materials as a result of the central metabolic role played by this dicarboxylic acid in the The Succinic Acid (Succinate) assay kit is suitable for the specific assay of succinic acid in wine, cheese, eggs, sauce and other food products. Succinic acid (or succinate) is found in all plant and animal materials as a result of the central metabolic role played by this dicarboxylic acid in the Citric Acid Cycle. Succinic acid concentrations are monitored in the manufacture of numerous foodstuffs and beverages, including wine, soy sauce, soy bean flour, fruit juice and dairy products (e.g. cheese).Product Description： Succinic acid is found in all plant and animal materials as a result of the central metabolic role played by this dicarboxylic acid in the Citric Acid Cycle. Succinic acid concentrations are monitored in the manufacture of numerous foodstuffs and beverages, including wine, soy sauce, soy bean flour, fruit juice and dairy products (e.g. cheese). The ripening process of apples can be followed by monitoring the falling levels of succinic acid. The occurrence of > 5 mg/kg of this acid in egg and egg products is indicative of microbial contamination. Apart from use as a flavouring agent in the food and beverage industries, succinic acid finds many other non-food applications, such as in the production of dyes, drugs, perfumes, lacquers, photographic chemicals and coolants. Preparation Instructions：Suitable for succinate determination in food, beverage, agricultural products, and other biological samples.Note for Content：The number of manual tests per kit can be doubled if all volumes are halved. This can be readily accommodated using the MegaQuantTM Wave Spectrophotometer (D-MQWAVE).Browse all of our organic acid assay kits.Principle：The Succinate Assay Kit provides a simple, one step assay for measuring succinate. In this assay succinate is converted to pyruvate which reacts with specific reagents and dye to form a colored product. The color intensity at 570 nm or fluorescencAdvantages：Extended cofactors stability. Dissolved cofactors stable for > 1 year at 4oC.Very competitive price (cost per test)All reagents stable for > 2 years as suppliedVery rapid reaction (even at room temperature)Mega-Calc™ software tool is available from our website for hassle-free raw data processingStandard includedSuitable for manual, microplate and auto-analyser formats... Read More |