| 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 | B665530 Component 50 T 200 T Storage B665530A Buffer RCL 125 mL 2×260 mL 2-8℃ B665530B Buffer GR 15 mL 50 mL RT B665530C Buffer GL 15 mL 50 mL RT B665530D Buffer GW1 (concentrate) 13 mL 52 mL RT B665530E Buffer GW2 (concentrate) 15 mL 50 mL RT B665530F Buffer GE 15 mL 60 mL RT B665530G B665530 Component 50 T 200 T Storage B665530A Buffer RCL 125 mL 2×260 mL 2-8℃ B665530B Buffer GR 15 mL 50 mL RT B665530C Buffer GL 15 mL 50 mL RT B665530D Buffer GW1 (concentrate) 13 mL 52 mL RT B665530E Buffer GW2 (concentrate) 15 mL 50 mL RT B665530F Buffer GE 15 mL 60 mL RT B665530G Proteinase K 1.25 mL 4×1.25 mL RT B665530H Spin Columns DM with Collection Tubes 50 sets 200 sets RTProduct IntroductionThis reagent kit is suitable for extracting total DNA, including genomic DNA, mitochondrial DNA, and viral DNA, from fresh or frozen whole blood (blood samples treated with anticoagulants such as citrate, EDTA, or heparin), plasma, serum, erythrocyte sedimentation rate brown layer, lymphocytes, cell-free body fluids, and other samples. This product can process 0.1-1 mL of whole blood with a maximum yield of 30% µ g. It can purify DNA with sizes ranging from 100 bp to 50 kb. The purified DNA has high yield and good quality, and can remove protein, pigment, lipid, and other inhibitory impurities to the maximum extent. It can be directly used for PCR, fluorescence quantitative PCR, enzyme digestion, and Southern Blot experiments.Self prepared reagent: anhydrous ethanol.Preparation and important precautions before the experiment:1. The sample should avoid repeated freeze-thaw cycles, otherwise it may result in smaller extracted DNA fragments and a decrease in extraction volume.2. This reagent kit can extract up to 0.1-1 mL of whole blood samples or 1 × 107 white blood cells.3.Before the first use, anhydrous ethanol should be added to Buffer GW1 and Buffer GW2 according to the instructions on the reagent bottle label.4. Before use, please check if there is any crystallization or precipitation in the Buffer GL. If there is any crystallization or precipitation, please incubate the Buffer GL in a 56 ℃ water bath and dissolve it again.5. The Buffer RCL in the reagent kit cannot be used again after being turbid.Operation steps:1. Sample processing: 1a When extracting 200 uL of blood sample, add the sample to the centrifuge tube (provided) and proceed directly to the next step of the experiment. 1b When the blood sample size is less than 200 µ When L, add Buffer GR to make up for 200 µ L. Proceed to the next step of the experiment. 1c When the blood sample size exceeds 200 µ When L is reached, add 1-2 times the volume of Buffer RCL, gently vortex or invert and mix well. Centrifuge at 12000 rpm (~13400 × g) for 1 minute and carefully discard the supernatant. If there is still red in the sediment, repeat the above steps once. Then add 200 to the precipitate µ Shake the buffer GR until thoroughly mixed before proceeding to the next step of the experiment. 1d If the processed blood sample is anticoagulant from poultry, birds, amphibians, or lower level organisms, its red blood cells are nucleated cells, and the blood sample size is 5-20 µ L. Can be added to Buffer GR to make up to 200 µ Follow up experiments will be conducted afterwards. Note: If downstream experiments are sensitive to RNA, 4 can be added µ L RNase A (100mg/mL) solution, shake for 15 seconds, and leave at room temperature for 5 minutes. RNase A reagent kit is not provided. If needed, you can order it separately from our company, item number: CW0601S.2. Add 20 to the above solution µ L Protein K, mix well.3. Add 200 µ Shake with L Buffer GL until thoroughly mixed. Note: Do not pre mix Protein K and Buffer GL.4.Incubate at 4.56 ℃ for 10 minutes, invert and mix several times during this time. Attention: The DNA production has reached its maximum after 10 minutes of incubation, and further extension of incubation time has no effect on DNA production and purity.5. Add 200 µ L anhydrous ethanol, invert and mix several times. Short centrifugation causes the liquid on the tube wall and wall cover to concentrate at the bottom of the tube.6. Add all the solution obtained in step 5 to the spin columns DM that have been loaded into the collection tube. If the solution cannot be added at once, it can be transferred multiple times. Centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.7. Add 500 to the adsorption column µ L Buffer GW1 (check if anhydrous ethanol is added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube. Note: If the extracted sample is the blood genome of species such as mice or monkeys that are difficult to remove heme, it is recommended to repeat step 7.8. Add 500 to the adsorption column µ L Buffer GW2 (check if anhydrous ethanol is added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube. Note: To further improve DNA purity, repeat step 8.9.Centrifuge at 9.12000 rpm for 2 minutes and discard the waste liquid from the collection tube. Place the adsorption column at room temperature for a few minutes to thoroughly air dry. Note: The purpose of this step is to remove residual ethanol from the adsorption column, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.)10. Place the adsorption column in a new centrifuge tube (provided by oneself) and add 50-200 to the middle of the adsorption column in the air µ L Buffer GE or sterilized water, leave at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 1 minute, collect DNA solution, and store DNA at -20 ℃. Note: 1) If downstream experiments are sensitive to pH or EDTA, they can be washed off with sterilized water. The pH value of the eluent has a significant impact on the elution efficiency. If water is used as the eluent, its pH value should be ensured to be between 7.0-8.5 (NaOH can be used to adjust the pH value of the water to this range). When the pH value is below 7.0, the elution efficiency is not high. 2) If the final concentration of DNA needs to be increased, the obtained DNA eluent can be added back to the adsorption membrane, left at room temperature for 2-5 minutes, and centrifuged at 12000 rpm for 1 minute. 3) Because DNA stored in water is affected by acidic hydrolysis, if long-term storage is required, it is recommended to elute with Buffer GE and store at -20 ℃... Read More | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | Format:2-ComponentEnzyme:Horseradish peroxidase | DescriptionThe Universal Coupling Kit makes particle-based immunoassays, lateral flow tests and biomolecule separation applications more flexible than ever before. It is the only kit that allows users to select and couple their choice of carboxylated particle with their chosen protein.Employing a DescriptionThe Universal Coupling Kit makes particle-based immunoassays, lateral flow tests and biomolecule separation applications more flexible than ever before. It is the only kit that allows users to select and couple their choice of carboxylated particle with their chosen protein.Employing a unique mechanism to immobilise proteins, Anteo′s advantages outweigh those of conventional covalent chemistries such as NHS/EDC or passive binding. This facilitates coupling of antibodies with ease, improved functionality and reproducibility, leading to better uniformity between experiments.Anteo′s Activation Reagent is water-based and replaces the dry chemicals you would use with the traditional NHS/EDC method. Our One-Step-Activation only takes one hour, and improves efficiency in terms of both time and cost. 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