| 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 | Cell proliferation detection is a basic experimental method to evaluate the health of cells, genotoxicity and the effect of antitumor drugs. The most accurate method to detect cell proliferation is the BrdU method. Edu detection kit is a revolutionary breakthrough of BrdU method. Edu (5-Cell proliferation detection is a basic experimental method to evaluate the health of cells, genotoxicity and the effect of antitumor drugs. The most accurate method to detect cell proliferation is the BrdU method. Edu detection kit is a revolutionary breakthrough of BrdU method. Edu (5-ethynyl-2 '- deoxyuridine) is a pyrimidine analog that integrates into the DNA duplex during DNA synthesis. Edu detection is based on the "click" reaction. A copper catalyzed azide reacts covalently with alkynes to form covalent bonds. In this kit, edu contains alkynes, Aladdin ® 488 / 555/594/647a azide dyes contain azide compounds. The edu labeling proliferation of click method is rapid and effective, and easy to use. BrdU method requires DNA denaturation (such as acid denaturation, thermal denaturation or digestion with DNase) to expose BrdU, so as to facilitate BrdU antibody binding; The edu method only needs paraformaldehyde fixation and Triton X-100 penetration to make the detection reagent enter the cells, and only a small amount of azide dye is needed to label the integrated edu very effectively. This kit contains all components required for edu method detection, and can be used for proliferation detection of cultured cells in vitro.Component:Product parameters：555/565 nm；Instruction： Experimental materials (self provided). 10 mM PBS, pH 7.2-7.6. 4% paraformaldehyde fixing solution (in PBS)Propensive reagent (0.5% Triton X -100 in PBS). 2 mg/mL glycine solution (in ddH2O). 3% BSA in PBS, pH 7.2-7.6. 1% BSA in PBS, pH 7.2-7.6. ddH2O. 96/24/12/6 well culture plate or dishFluorescence microscopy detection method1. Cell cultureTake logarithmic growth stage cells and inoculate them into a 96 well plate with 4 × 103-1 × 105 cells per well (the number and density of cells can be adjusted according to cell size, growth rate, and specific requirements of experimental treatment), and culture until normal growth stage.2. Drug treatmentPerform various drug treatments according to experimental needs.3. EdU marking(1) Dilute EdU solution (component A) in a certain proportion with complete cell culture medium to an appropriate concentration, then add it to the cells and mix well; Set up a negative control group without EdU treatment.Note: The labeling concentration of EdU needs to be adjusted according to cell type, and it is recommended to explore with an initial concentration of 10 µ M. In the pre experiment, it is recommended to set an EdU concentration gradient, which can be referred to in Tables 2 and 3.(2) Incubate in a cell culture incubator for 2 hours.Note: The optimal incubation time is related to the cell cycle. Most tumor cell lines can use a 2-hour incubation time, as shown in Appendix 2. The concentration of EdU is related to the incubation time, and high concentrations, such as 10-50, should be used for short-term incubation (<2 hours) µ M; Long term incubation (>24 hours) should use low concentrations, such as 1-10 µ M; You can also refer to Appendix 3.4. Cell fixation and permeation promotionNote: For experiments that require cell surface antigen labeling, it can be considered to wash the cells twice with a 3% BSA washing solution after completing EdU incubation, before cell fixation and permeation promotion.(1) After incubation, remove the culture medium. Wash cells twice with 1X PBS for 5 minutes each time to remove EdU residues that have not been incorporated into DNA. Cells with weak adhesion can reduce cleaning intensity. Join 50 µ After incubating at room temperature for 20 minutes with 4% paraformaldehyde fixative, remove the fixative.(2) Add 50 to each hole µ L 2 mg/mL glycine solution, incubate at room temperature for 5 minutes, and neutralize the remaining fixed solution.(3) At a rate of 100 per hole µ Wash cells twice with 3% BSA.(4) Remove the washing solution and add 100 µ L 0.5% Triton X -100, incubate at room temperature for 10 minutes.5. EdU detectionNote: Each sample in this reference step uses 100 µ The working fluid of L can be adjusted by users according to their own sample situation.(1) Prepare 1 x Click iT EdU reaction buffer (component C): Dilute component C 10 times with ddH2O.(2) Configure 5 x Click iT EdU buffer additives (component E): add 300 µ Mix L of ddH2O into a 30 mg E component tube (final concentration of 100 mg/mL) until completely dissolved. After use, the remaining storage solution is stored at -20 ℃ and can be stored for one year. Once the solution turns brown, it indicates that the active ingredients have degraded and cannot be reused.Note: Different specifications of component E are dissolved in ddH2O according to this ratio, and prepared into a 5 x storage solution for future use.(3) Prepare 1 x Click iT EdU buffer additive: Dilute 5 x Click iT EdU buffer additive with ddH2O to 1 x, and the solution should be prepared and used immediately.(4) Prepare Click it working solution according to Table 1.Table 1 Click it working fluidReaction componentsTaking the sample size of 10 holes as an example1×Click-iT EdU Reaction buffer855 µLCuSO4 (Component D)40 µLYF® 488/555/594/647A Azide(Component B)5 µL1×Click-iT EdU Buffer additives100 µLTotal volume1 mL(5) Remove penetration enhancer, 100 per well µ Wash twice with 3% BSA washing solution of L.(6) Add 100 to each hole µ L Click iT working solution, evenly covering cells.(7) Incubate at room temperature in dark for 30 minutes.(8) Remove Click-iT working fluid and add 100 µ After washing cells twice with 3% BSA, remove the washing solution and add 100 µ L PBS keeps cells moist. If there are no other special requirements, photography analysis can be carried out.6. DNA re staining (optional)(1) Using 100 µ Wash the cells once with PBS and remove the washing solution.(2) Dilute Hoechst 33342 (component F) 2000 times with PBS.(3) Add 100 to each hole µ Incubate L 1 x Hoechst 33342 solution at room temperature in dark for 15-30 minutes.(4) Remove Hoechst 33342 solution and use 100 µ Wash cells twice with PBS.7. Imaging and analysisIt is recommended to take fluorescence microscopy photos immediately after staining is completed for observation; If conditions permit, please store in a dark and moist environment at 4 ° C for 3 days before taking photos. Flow cytometry detection method1. Cell cultureInoculate 1 × 105~3 × 106 cells per well into a 6-well plate.2. Drug treatmentPerform various drug treatments according to experimental needs.3. EdU labeled cells(1) Dilute EdU solution (component A) in a certain proportion with complete cell culture medium to an appropriate concentration, then add it to the cells and mix well; Set up a negative control group without EdU treatment.Note: The labeling concentration of EdU needs to be adjusted according to cell type, and it is recommended to explore with an initial concentration of 10 µ M. In the pre experiment, it is recommended to set an EdU concentration gradient, which can be referred to in Tables 2 and 3.(2) Incubate in a cell culture incubator for 2 hours. The time of EdU incubation of cells can be directly used as an indicator for measuring cell DNA synthesis, and the choice of time point and incubation time depend on the cell growth rate. Pulse labeled cells incubated with brief EdU can be used to study cell cycle dynamics.Note: The optimal incubation time is related to the cell cycle. Most tumor cell lines can use a 2-hour incubation time, as shown in Appendix 2. The concentration of EdU is related to the incubation time, and high concentrations such as 10-50 should be used for short-term incubation (<2 hours) µ M; Long term incubation (>24 hours) should use low concentrations, such as 1-10 µ M; You can also refer to Appendix 3.4. Cell fixation and permeation promotionNote: For experiments that require cell surface antigen labeling, it can be considered to wash cells twice with 1% BSA after completing EdU incubation, before cell fixation and permeation promotion.(1) After incubation, collect cells, add 1 mL of PBS to each tube to clean the cells, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant to remove EdU residue that has not been added to DNA.(2) Add 1 mL of 4% paraformaldehyde fixative to each tube to resuspend cells.(3) Incubate at room temperature for 20 minutes, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant.(4) Add 1 mL of 2 mg/mL glycine to each tube and incubate for 5 minutes. Neutralize the remaining fixed solution, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Add 1 mL of PBS to each tube for cleaning once, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant.(5) Add 1mL of 0.5% Triton X-100 osmotic enhancer to each tube and resuspend cells. Incubate at room temperature for 10 minutes.5. EdU detectionNote: For 6-well plate samples, reference can be made to 1 mL of working solution per well. Users can adjust the dosage according to their own sample situation.(1) Prepare 1 x Click iT EdU reaction buffer: Dilute component C 10 times with ddH2O.(2) Prepare 5 x Click iT EdU buffer additives (component E): Add 300 µ L ddH2O to 30 mg of component E in a test tube (final concentration 100 mg/mL), mix well until completely dissolved. After use, the remaining storage solution is stored at -20 ℃ and can be stored for one year. Once the solution turns brown, it indicates that the active ingredients have degraded and cannot be reused.Note: Different specifications of component E are dissolved in ddH2O according to this ratio to form 5 x storage solution for future use.(3) Prepare 1 x Click iT EdU buffer additive: Dilute 5 x Click iT EdU buffer additive storage solution with ddH2O to 1 x, and the solution should be prepared and used immediately.(4) Prepare Click it working solution according to Table 2.Table 2 Click it working fluidReaction componentsVolume of liquid required for a single reaction1×Click-iT EdU Reaction buffer875 µLCuSO4 (Component D)20 µLYF® 488/555/594/647A Azide(Component B)5 µL1×Click-iT EdU Buffer additives100 µLTotal volume1 mL(5) Soak at 1000 rpm for 5 minutes, discard the supernatant, remove the enhancer, add 1mL of 1% BSA washing solution to each tube and wash twice. Soak at 1000 rpm for 5 minutes, discard the supernatant.(6) Add 1 mL of Click iT working solution to each tube and mix well.(7) Incubate at room temperature in dark for 30 minutes.(8) Soak at 1000 rpm for 5 minutes, discard the staining reaction solution, add 1% BSA to each tube to wash the cells twice, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend the cells again with 1 mL of 1% BSA (the volume of resuspend cells can be adjusted according to the number of cells), and detect with a flow cytometer.Note: If other biomarker tests are required, please refer to step 4.6. Intracellular antigen labeling (optional steps)(1) Add antibody working solution and mix well.(2) Under dark conditions, incubate antibodies at appropriate temperature and time.7. Flow detection and analysis:(1) It is recommended to conduct flow cytometry testing immediately after dyeing is completed; If conditions are limited, please store in a dark place at 4 ℃ for testing, but it should not exceed 3 days.(2) It is recommended to test the number of cells up to one million levels as much as possible. If the number of cells is small, the number of cells tested can be adjusted to 100000 levels starting from the experiment. For cases where the cell yield is too low (just to the level of ten thousand), it may not be conducive to making a flow chart. Therefore, the cleaning frequency in step 5 (8) can be appropriately reduced.Matters 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 during experimental operation to slow down fluorescence quenching. 3. click it edu buffer additive solution should be prepared and used immediately to ensure the best results. 4. for your safety and health, please wear experimental clothes and disposable gloves.Scope of application：Cell proliferation detection (cell imaging flow universal)... Read More | Hydrogen peroxide, a reactive oxygen species produced through the metabolism of molecular oxygen, serves as both an intracellular signaling messenger and a source of oxidative stress. Hydrogen peroxide is generated in cells via multiple mechanisms such as the NOX-mediated ROS production by Hydrogen peroxide, a reactive oxygen species produced through the metabolism of molecular oxygen, serves as both an intracellular signaling messenger and a source of oxidative stress. Hydrogen peroxide is generated in cells via multiple mechanisms such as the NOX-mediated ROS production by neutrophils and macrophages (respiratory burst) or by the dismutase of superoxide anions produced as a result of electron leak during mitochondrial respiration. Abnormal hydrogen peroxide production contributes to oxidative cell damage and the progression of diseases such as asthma, atherosclerosis, osteoporosis, and neurodegeneration.Intracellular hydrogen peroxide assay kit has been used to measure intracellular hydrogen peroxide levels... Read More | DescriptionThe plasma protein lecithin:cholesterol acyltransferase (LCAT) catalyzes the transfer of an acyl group from the sn2 position of phosphatidylcholine to the 3-hydroxyl group of cholesterol forming cholesteryl ester. This activity occurs on the surface of high density lipoprotein (HDL) and DescriptionThe plasma protein lecithin:cholesterol acyltransferase (LCAT) catalyzes the transfer of an acyl group from the sn2 position of phosphatidylcholine to the 3-hydroxyl group of cholesterol forming cholesteryl ester. This activity occurs on the surface of high density lipoprotein (HDL) and the cholesteryl esters formed by LCAT are incorporated into the core of HDL.Preparation instructionsSuitable for high-throughput screening, mechanism of action studies and structureactivity relationship (SAR) work of LCAT in plasma and serumPrincipleThe LCFC-LCAT Acyltransferase Activity Assay is a fluorometric assay useful for measuring the acyltransferase activity of LCAT in serum or plasma. The method detects changes in LCAT free cholesterol (LCFC) levels in the sample without the use of c... Read More | Product contentY666144Component50 TStorageY666144ABuffer P115 mLRTY666144BBuffer P215 mLRTY666144CBuffer N320 mLRTY666144DBuffer PS15 mLRTY666144EBuffer PB10 mLRTY666144FBuffer PW (concentrate)10 mLRTY666144GBuffer EB10 mLRTY666144HGlass Beads2 gRTY666144IRNase A (10mg/mL)150 µLRTY666144JSpin Product contentY666144Component50 TStorageY666144ABuffer P115 mLRTY666144BBuffer P215 mLRTY666144CBuffer N320 mLRTY666144DBuffer PS15 mLRTY666144EBuffer PB10 mLRTY666144FBuffer PW (concentrate)10 mLRTY666144GBuffer EB10 mLRTY666144HGlass Beads2 gRTY666144IRNase A (10mg/mL)150 µLRTY666144JSpin Columns DM with Collection Tubes50 setsRTProductsThis kit is improved on the basis of common alkaline lysis method, the glass beads can effectively break the yeast cell wall, the new silica matrix membrane and buffer system can efficiently and specifically bind the plasmid DNA, and at the same time can maximize the removal of proteins and other impurities, the whole process is convenient and fast, no need to use toxic and harmful reagents, and can be processed at the same time for multiple samples. In addition to yeast cells, it can also be used in E. coli. Plasmid DNA extracted with this kit can be used in various molecular biology experiments, such as ligation, transformation, sequencing and library screening.Self-contained reagents: β-mercaptoethanol, anhydrous ethanol.Pre-experiment Preparation and Important Notes1. All components can be stably stored in dry, room temperature (15-30℃) environment for 1 year, the adsorption column can be stored at 2-8℃ for a longer period of time, and Buffer P1 with RNase A can be stably stored at 2-8℃ for 6 months.2. Before the first use, add all the RNase A solution to Buffer P1, mix well, and store at 2-8℃.3. Anhydrous ethanol should be added to Buffer PW before first use according to the instructions on the reagent bottle label.4. Before use, please check whether Buffer P2 and Buffer N3 are crystallized or precipitated. If there is any crystallization or precipitation phenomenon, it can be clarified by taking a water bath at 37℃ for a few minutes to restore the clarity.5. Be careful not to touch Buffer P2 and Buffer N3 directly, and tighten the lid immediately after use.6. The amount of plasmid extracted is related to the yeast strain, plasmid copy number, culture conditions, etc. Usually, yeast plasmid copy number is very low, which is difficult to be detected by electrophoresis or spectrophotometer method.Procedure1. Take 1-5 ml of yeast culture (maximum 5×107 yeast cells, generally for Saccharomyces cerevisiae OD = 1.0, equivalent to 1-2×107 cells/ml) and add it to a centrifuge tube (self-provided), centrifuge for 30 seconds at 12,000 rpm (~13,400×g), collect the bacterial precipitate, and aspirate as much as possible to discard the supernatant.2. Add 250µl Buffer P1 to the bacterium (please check if RNase A has been added first) and resuspend the precipitate.3. Add 40mg of Glass Beads to the above mixture and vortex and shake for 10 minutes.4. Add 250 µl of Buffer P2 to the centrifuge tube, mix gently by turning up and down 6-8 times, and let stand at room temperature for 5-10 minutes, at which time the bacterial solution should become clear and viscous.Note: Mix gently, do not shake violently, so as not to interrupt the genomic DNA, resulting in genomic DNA fragments mixed in the extracted plasmid. If the solution does not become clear, it suggests that the amount of bacteria may be too large and the lysis is not complete, and the amount of bacteria should be reduced.5. Add 350 µl of Buffer N3 to the centrifuge tube and immediately mix gently up and down 6-8 times, at which point a white flocculent precipitate appears, and centrifuge at 12,000 rpm for 20 minutes.Note: Buffer N3 should be mixed immediately after addition to avoid localized precipitation.6. Column Equilibration: Add 200 µl of Buffer PS to the Spin Columns DM in the collection tube, centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid from the collection tube, and place the column back into the collection tube.7. Add the supernatant from step 5 to the adsorbent column that has been loaded into the collection tube, taking care not to aspirate the precipitate.Note: The maximum volume of the adsorption column is 750 µl, and the solution is passed through the column in 2 times.8. Centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid in the collection tube and place the adsorption column back into the collection tube.9. Add 150 µl Buffer PB to the adsorbent column, centrifuge at 12,000 rpm for 1 min, pour off the waste liquid in the collection tube, and put the adsorbent column back into the collection tube.10. Add 750 µl Buffer PW to the adsorption column (please check that anhydrous ethanol has been added first), centrifuge at 12,000 rpm for 1 minute, and pour off the waste liquid in the collection tube.11. Place the column back into the recovery collection tube and centrifuge at 12,000 rpm for 2 minutes, pouring off the waste liquid. Leave the column at room temperature for several minutes to dry thoroughly.Note: The purpose of this step is to remove residual ethanol from the adsorption column; ethanol residue can interfere with subsequent enzymatic reactions (digestion, PCR, etc.).12. Place the adsorbent column in a new centrifuge tube, add 50-100 µl of Buffer EB to the center of the adsorbent membrane dropwise, let it stand at room temperature for a few minutes, centrifuge at 13,000 rpm for 1 minute, and collect the plasmid solution into the centrifuge tube. Store the plasmid at -20°C.Attention:1) To increase the recovery efficiency of the plasmid, the resulting solution can be reintroduced into the adsorbent column, left at room temperature for a few minutes, centrifuged at 13,000 rpm for 1 minute, and the plasmid solution collected into a centrifuge tube.2) When the plasmid copy number is low or >10 kb, Buffer EB is preheated at 65-70°C in a water bath, which can increase the extraction efficiency.3) Usually yeast plasmids have very low copy number and are difficult to detect by electrophoresis or spectrophotometry. If the extracted plasmid is to be used in the next step of the experiment, it is usually recommended to use 1-5µl of the plasmid as PCR template, and 5-10µl of the plasmid for transformation of E. coli.4) Commercial high transformation efficiency receptor cells should be used for transformation of E. coli... Read More |