| Description | Hydrogen peroxide (H₂O₂) is the most common reactive oxygen species (ROS) in living organisms. It is a by-product of active oxygen metabolism, primarily produced by enzymes like SOD and XOD, and degraded by enzymes such as CAT and POD. H₂O₂ is not only a significant ROS but Hydrogen peroxide (H₂O₂) is the most common reactive oxygen species (ROS) in living organisms. It is a by-product of active oxygen metabolism, primarily produced by enzymes like SOD and XOD, and degraded by enzymes such as CAT and POD. H₂O₂ is not only a significant ROS but also a hub for the interconversion of reactive oxygen species. On one hand, H₂O₂ can directly or indirectly oxidize biological macromolecules like nucleic acids and proteins within cells, damaging cell membranes and thereby accelerating cellular aging and disintegration. On the other hand, H₂O₂ is also a key regulatory factor in many oxidative stress responses. It can activate factors like NF-κB, and these H₂O₂-related signaling pathways are associated with many diseases such as asthma, inflammatory arthritis, arteriosclerosis, and neurodegenerative diseases. H₂O₂ is also closely related to processes like cell apoptosis and proliferation.Detection Principle: H₂O₂ oxidizes ferrous ions (Fe²⁺) to ferric ions (Fe³⁺). The Fe³⁺ then forms a purple complex with xylenol orange in a specific solution. The absorbance at 580 nm is directly proportional to the H₂O₂ concentration, allowing for the quantification of H₂O₂ levels.Detection Range: 1-100 µMSensitivity: 1 µMApplicable Samples: Animal/plant tissues, cells, bacteria, serum (plasma), urine.H1492752Component96T480TStorageH1492752AReaction Buffer5 mL25 mL-20℃. Store in the dark.H1492752BH₂O₂ Standard (1M)0.1 mL0.1 mL-20℃. Store in the dark.H1492752CAssay Buffer (10×)13 mL65 mL2-8℃Please check the quantity of each component before the experiment.An additional 10% of each component is provided beyond the specified volume for standard curve preparation or preliminary experiments.User-Provided Instruments and ReagentsTypeNameNotesInstrumentMicroplate ReaderCapable of measuring absorbance at 580 nm.Consumables96-well Microplate / Ultrafiltration tubesStandard transparent plate / 10 kDa MWCOReagentsPBS (pH 7.4) / Deionized Water / 30% ZnSO₄ solutionFor washing cells/bacteria / Reagent preparation / Protein removalOthersHomogenizer (for tissue samples), incubator, ice bucket, low-temperature centrifuge, adjustable pipettes and tipsUsing a multichannel pipette for large-scale detection can improve efficiency.Experimental Procedure1. Reagent PreparationReagent NameReagent PreparationPrecautionsReaction BufferReady-to-use; equilibrate to room temperature before use.Protect from light during the experiment; aliquot and store at -20°C in the dark.H₂O₂ Standard (1M)Ready-to-use; equilibrate to room temperature before use.Protect from light during the experiment; aliquot and store at -20°C in the dark.Assay Buffer (1×)Dilute the 10× Assay Buffer 1:10 with deionized water before use; equilibrate to room temperature.The diluted buffer can be stored at 4°C for at least 2 months. Used for diluting H₂O₂ standard and samples.2. Standard PreparationStandard Curve Setup:First, prepare a 2 mM H₂O₂ Standard: Dilute 2 µL of the 1M H₂O₂ Standard with 998 µL of Assay Buffer (1×).Then, prepare a 100 µM H₂O₂ Standard: Dilute 50 µL of the 2 mM H₂O₂ Standard with 950 µL of Assay Buffer (1×).Using the 100 µM H₂O₂ Standard, prepare further dilutions as shown in the table below.Prepare fresh standard solutions for each experiment.Prepared standards must be used within 4 hours.If the sample is a cell suspension, it is recommended to prepare the H₂O₂ standards using the culture medium.Standard Working Solution100µM Standard (µL)Assay Buffer (1×) (µL)Concentration (µM)1200010021001005034016020420180105101905641962721981Blank020003. Sample PreparationNote: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 1 month. When ready for the experiment, thaw samples on ice. Note that this may affect sample stability, and results might be lower than expected. The following substances interfere with detection and should be avoided in samples: Ferric salts, iron salts, sucrose, glucose, ascorbic acid, SDS (>0.2%), sodium azide.3.1 Animal Tissues:Wash the tissue with cold PBS to remove as much blood as possible. Blot dry, weigh 0.1 g, and add 1 mL of pre-cooled Assay Buffer (1×). Homogenize the sample on ice. Centrifuge at 10000 g, 4°C for 5 min. Collect the supernatant and keep on ice for detection.3.2 Plant Tissues:Weigh approximately 0.1 g of sample, add 1 mL of pre-cooled Assay Buffer (1×), and grind. Disrupt by ultrasound on ice (power 20% or 200 W, ultrasonicate for 3 s, interval 7 s, repeat 30 times). Centrifuge at 10000 g, 4°C for 5 min. Collect the supernatant and keep on ice for detection.3.3 Cells/Bacteria:Collect 5×10⁶ cells or bacteria. Wash with cold PBS, then add 1 mL of pre-cooled Assay Buffer (1×). Homogenize on ice or disrupt by ultrasound on ice (power 20% or 200 W, ultrasonicate for 3 s, interval 7 s, repeat 30 times). Centrifuge at 10000 g, 4°C for 5 min. Collect the supernatant and keep on ice for detection.3.4 Plasma, Serum, and Urine (and other biological fluids):Remove proteins and use the supernatant. Protein removal methods:Use a 10 kDa ultrafiltration tube: filter and collect the filtrate.Mix sample : 30% ZnSO₄ solution = 20 : 1, vortex, then centrifuge at 10000 g, room temperature for 5 min, and collect the supernatant.4. Assay Steps4.1 Microplate Reader Preparation: Preheat for at least 30 minutes, set wavelength to 580 nm.4.2 Assay System Setup:ReagentStandard Well (µL)Test Well (µL)Standard (various conc.)600Sample060Reaction Buffer40404.3 Mix the reaction system thoroughly and incubate at 37°C for 10 minutes.4.4 Absorbance Measurement: Read the absorbance at 580 nm, recorded as A blank, A standard, and A test. 5. Result CalculationThe following provides both the derived formula and the simplified calculation formula, which are completely equivalent.5.1 Data ProcessingCalculate ΔA standard = A standard - A blank, ΔA test = A test - A blank. 5.2 Standard Curve PlottingPlot the standard curve with standard concentration as the y-axis and ΔA <sub> standard </sub> as the x-axis. Substitute ΔA <sub> test </sub> into the equation to obtain the y value (µM).5.3 Sample H₂O₂ Concentration Calculation(1) Based on sample mass:H₂O₂ Content (nmol/g fresh weight) = y × V sample ÷ (W × V sample ÷ V total ) × n = y ÷ W × n(2) Based on cell or bacterial count:H₂O₂ Content (nmol/10⁴ cells) = y × V sample ÷ (500 × V sample ÷ V total ) × n = y ÷ 500 × n(3) Based on liquid volume:H₂O₂ Content (nmol/mL) = y × V sample ÷ V sample × n = y × nParameter Description:1 µM = 1 nmol/mL;V sample : Volume of sample added;V total : Volume of Assay Buffer (1×) added, 1 mL;n: Sample dilution factor;W: Sample mass, g;500: Cell or bacterial count, in units of 10⁴.6. Result PresentationTypical Standard Curve: y = 207.21x + 1.4921, R² = 0.9988Example-1: 0.1 g of corn tissue was processed and assayed according to the procedure using a 96-well plate.Measured: ΔA test = A test - A blank = 0.278 - 0.048 = 0.230Substituting into the standard curve gives y = 49.15 µM.Calculated based on sample mass:H₂O₂ Content (nmol/g) = y ÷ W × n = 491.5 nmol/g.Precautions1. It is recommended to perform preliminary experiments using 2-3 samples expected to have significant differences before formal testing.2. This kit is compatible with spectrophotometer detection. Adjust the preparation volume of detection reagents proportionally according to the spectrophotometer's requirements.3. 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.4. 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.5. This product is for scientific research use only. Not intended for clinical diagnosis.Frequently Asked QuestionsQ: What should I do if the sample ΔA <sub> test </sub> is too high or too low?A: If the sample ΔA test is greater than the ΔA standard of the 100 µM standard, the H₂O₂ content in the sample is too high. Dilute the sample appropriately with Assay Buffer (1×) (multiply by the dilution factor in the calculation). If the sample ΔA test is less than 0.005, increase the sample amount... Read More | Product Descriptionalpha-L-fucoside fucohydrolase, alpha-L-fucosidase, alpha-(1-3,4) fucosidaseAlpha (1-3,4) Fucosidase The enzyme is very efficient and recognises α1-3,4 fucosylated glycans (e.g. Lewis X/A epitopes, including their sialylated counterparts) and hydrolyses terminal α1-3 andProduct Descriptionalpha-L-fucoside fucohydrolase, alpha-L-fucosidase, alpha-(1-3,4) fucosidaseAlpha (1-3,4) Fucosidase The enzyme is very efficient and recognises α1-3,4 fucosylated glycans (e.g. Lewis X/A epitopes, including their sialylated counterparts) and hydrolyses terminal α1-3 and α1-4 fucosyl linkages in these substrates without the need to remove sialic acid moieties.For removing core fucose linked α-(1-6) to the core GlcNAc of a GlcNAc-GlcNAc disaccharide structure we recommend our Alpha-(1-6) Fucosidase.• Non-sialidase dependant hydrolysis of antennary fucose moieties• Effective on both glycopeptides and free glycans• Highly specific (α1-3,4 fucosylated glycans)• Kit includes enzyme plus reaction buffer.• Sufficient for up to 50 samplesα(1-3,4) Fucosidase is useful for:nbsp;nbsp;Fucose linkage determinationnbsp;nbsp;Deglycosylating glycoproteins with Lewis structuresContentsAlpha-(1-3,4)-Fucosidase – 200 mM citrate buffer pH 6 containing 250 mM NaCl5x Reaction Buffer – 250 mM sodium phosphate pH 6... 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 | The fluorescent dye PKH67 is suitable for conventional cell membrane labeling. It is a green fluorescent dye that can track cells in vitro and in vivo. It labels cells by binding to the lipid components of the membrane structure. PKH67 has low cytotoxicity, low fluorescence background, high fat The fluorescent dye PKH67 is suitable for conventional cell membrane labeling. It is a green fluorescent dye that can track cells in vitro and in vivo. It labels cells by binding to the lipid components of the membrane structure. PKH67 has low cytotoxicity, low fluorescence background, high fat solubility, can easily penetrate cell membranes, and has strong and stable green fluorescence. PKH67-labeled cells can be used for in vitro and in vivo proliferation studies, and have the function of not staining neighboring cells. In the process of cell division and proliferation, the fluorescence intensity of PKH67 will gradually decrease as the cells divide. The labeled fluorescence can be evenly distributed to the two sub-generation cells, so its fluorescence intensity is half that of the parent cell. According to this feature, It can be used to detect cell proliferation, cell cycle estimation and cell division, etc. The fluorescence of PKH67-labeled cells is very uniform, and the fluorescence distribution of sub-generation cells after division is also more uniform. In the process of cell division and proliferation, PKH67-labeled fluorescence can be evenly distributed between the two sub-generation cells, and the fluorescence intensity becomes half of that of the parent cell. According to the difference in fluorescence intensity, the undivided cells can be detected by flow cytometry. One time (1/2 the fluorescence intensity), the second time (1/4 the fluorescence intensity), three times (1/8 the fluorescence intensity), and more divisions of cells. PKH67 can detect splits up to six times or even more. In addition to the detection of cell proliferation, PKH67 can also be used for in vitro tracking of cells. After labeling, the fluorescence expression is stable in the cell, and the positive labeling rate is over 98%. The labeled cells have good morphology, which can effectively observe the cells in vitro. Induce differentiation; or inject labeled cells into the body, it can effectively show the migration and differentiation of transplanted cells in living tissues. PKH67-labeled cells can be used for in vivo observation for as long as several weeks. It is often used for in vivo cell detection experiments and experiments to observe long-term cell activity using fluorescence electron microscope. PKH67 is less toxic and does not affect cell proliferation. This method is simple to operate, does not use radioactive isotopes, and poses no safety hazards. You can get the desired experimental data faster, more accurately and more safely.Due to the longer length of the charcoal tail, internal studies have shown that PKH67 is less transferred between cells than PKH2. In in vivo studies using PKH1 and PKH2, the fluorescence intensity will slowly lose. Since this is a behavioral characteristic of green cell linker dye rather than red cell linker dye, PKH67 will have similar properties. The correlation between the in vitro cell membrane retention of non-dividing cells and the in vivo fluorescence half-life reveals that the in vivo fluorescence half-life of PKH67 is 10-12 days. Other green cell linker dyes with similar half-lives have been used to monitor the transport of lymphocytes and macrophages in the body within one to two months. The results indicate that PKH67 can also be used for medium-term in vivo tracking studies.The dye can stably bind to the lipid region of the cell membrane and emit fluorescence, and is mainly used for cell labeling in vitro, cell proliferation research in vitro, and cell tracing research in vivo and in vitro. The fluorescence half-life of PKH67 in vivo is 10-12 days. Compared with PKH-67, PKH-26 has a longer half-life, and the half-life of PKH26 labeled on rabbit red blood cells is more than 100 days. Especially suitable for in vitro proliferation research and long-term in vivo cell tracking research. After PKH67 labels the cells, flow cytometry is usually used for cell proliferation detection.Kit components0.1ml kits: P266290A-0.1ml P266290B-10ml1ml kits: P266290A-1ml P266290B-60mlDyes with A suffix and diluents with B suffix are used togetherPKH67 labeled cells show green fluorescence, the fluorescence wavelength: λex=490 nm, λem=502 nm.Storage conditions: -20℃ protected from light, valid for 1 yearPrecautions●Staining concentration varies according to the type of cell and the number of cells in each well.● The prepared PKH67 mother liquor is very easy to dissolve. It is recommended to store in aliquots and freeze-dry at ≦-20℃.● PKH67 working solution should be prepared for immediate use, and cannot be prepared in advance, because PKH67 will decompose due to the absorption of water and affect the dyeing effect.● PKH67 is easily decomposed and will deteriorate quickly in the water solution. Please avoid contact with water during use of mother liquor. The working fluid is in contact with the water during the process of labeling the cells within the permitted time range.● PKH67 fluorescent dye is a DMSO solution. It will solidify and stick to the bottom, wall or cap of the tube at a lower temperature such as 4℃ and ice bath. After being taken out of the refrigerator, it will return to room temperature and become After the liquid is in the state, remove the cap from the bottom of the tube. It can be used after it has completely melted in a 37°C water bath.● The number of generations or time that can be traced after different cell types are marked is quite different. Please make a test based on the actual situation or reference documents.Instructions1. Staining solution preparation:(1) Take out the PKH67 reagent from the refrigerator, let it stand for a few minutes to room temperature, or after a 37°C water bath, leave the tube containing PKH67, and be sure to leave the tube for a few minutes before opening the lid to allow the reagent to fully fall into the tube The lid can only be opened after the bottom.(2) According to the number of cell samples to be tested, dilute the probe 10 times with the diluent, and then use a suitable solution (such as non-clear medium, HBSS or PBS) to dilute the PKH67 mother liquor 25 times to prepare a stain Work fluid. The best working solution concentration should be adjusted according to different cells and your own experimental system. Generally, the cells can be diluted 250 times according to the final concentration of the mother liquor in the kit. Some cells may need to increase the concentration appropriately.2. Cell staining(1) Resuspend the prepared cells to be tested in 100µl of staining solution to a cell concentration of about 107/ml. You can also perform in-situ staining, as long as the staining solution is enough to cover the cells.(2) Culture the cells at 2～8℃ for 15～30 minutes. The best culture time is different for different cells.It is recommended to incubate the labeled cells in the staining solution at 37°C for 5 minutes, and then at 4°C for 15 minutes.Low-temperature incubation can reduce the endocytosis of the dye by the cells, help the dye to label the plasma membrane, and reduce the possibility of the dye localizing to cytoplasmic vesicles.(3) After separation, remove the supernatant, collect the cells, wash the cells 1-2 times with PBS or non-clear medium, and finally add PBS or non-clear medium to resuspend the cells.(4) Take 500µl of cell suspension and test with flow cytometer. Ex/Em=490/502nm.(5) Subsequently, the cells can be cultured according to the normal culture method.(6) The labeling effect can be directly observed under a fluorescence microscope, or the cell proliferation can be detected by a flow cytometer after an appropriate period of culture, or used for cell fluorescence traces for other specific experimental purposes... Read More | Product contentComponentY665957-1mlY665957-5ml2×GoldStar Probe Mixture1 ml5×1 mlProbe Primer Mix300 µl5×300 µlHuman DNA Standard（100 ng/µl）100 µl5×100 µl50×High ROX40 µl200 µlProduct IntroductionThis product is a real-time Product contentComponentY665957-1mlY665957-5ml2×GoldStar Probe Mixture1 ml5×1 mlProbe Primer Mix300 µl5×300 µlHuman DNA Standard（100 ng/µl）100 µl5×100 µl50×High ROX40 µl200 µlProduct IntroductionThis product is a real-time fluorescence quantitative PCR kit for detecting the concentration of human male Y chromosome, including carefully optimized PCR reaction solution, primer mixture and standards, especially suitable for the quantitative detection of precious and micro DNA samples. The kit adopts a new efficient and fast hot-start amplification enzyme GoldStar Taq DNA Polymerase, which effectively avoids non-specific amplification caused by non-specific binding of primers and templates or primer dimerization at room temperature. This product realizes accurate quantification of Y chromosome and can be applied in various fields such as genetic mapping, species polymorphism research, disease gene localization, paternity testing and forensic analysis.ROX dye is used to correct the fluorescence signal error generated between wells of a quantitative PCR instrument, and is generally used in Real Time PCR amplifiers from ABI, Stratagene, and other companies. The excitation optics vary from instrument to instrument, so the concentration of ROX dye must be matched to the corresponding fluorescence quantitative PCR instrument.Instruments that do not require ROX calibration: Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96, etc.Instruments requiring Low ROX calibration: ABI Prism7500/7500 Fast, QuantStudio®3 System, QuantStudio®5 System, QuantStudio®6 Flex System, QuantStudio®7 Flex System, ViiA 7 System, Stratagene Mx3000/Mx3005P, Corbett Rotor Gene 3000, and others.Instruments requiring High ROX calibration: ABI Prism7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, etc.Note: High Rox and Low Rox are formulated as described in Method of Use 3.Scope of applicationThis product is suitable for quantitative testing of male Y chromosome DNA in scientific research, clinical, forensic medicine and paternity testing.Usage1. Amplification template preparationThe library samples to be detected were diluted with TE (10 mM Tris-Cl, pH 8.0, 1 mM EDTA), and the concentration after dilution was as close as possible to the range of 0.05-10 ng/µL. 4°C on ice was set aside.2. Standard dilution: according to the following table, firstly dilute Human DNA Standard (100ng/uL) with TE to make 5 standards of different concentrations according to the table below. 10ng/µL of DNA Standard 1 (Std.1) can be stored stably at -20℃ for 1 month; Std2-5 can only be used on the same day, and should be placed at 4℃ or on ice when not in use for the time being after preparation. When Std2-5 are not used temporarily after preparation, they should be stored at 4℃ or on ice.Standard sampleCorresponding concentration(ng/µl)Minimum Dilution Volume (Unit：µl）Std.11010 [100 ng/µl DNA Standard]+ 90 TEStd.22.520 [Std. 1] +60 TEStd.30.62520 [Std. 2] +60 TEStd.40.1562520 [Std. 3] +60 TEStd.50.039062520 [Std. 4] +60 TE3. qPCR reaction system preparationBefore preparation, the cryopreserved reagents to be used were completely melted and mixed by inverting several times, then centrifuged briefly and prepared. Standards and templates were diluted as described above and prepared.The base reaction system for 20 µL was as follows:Reagent20 µl Reaction system2×GoldStar Probe Mixture10 µlProbe Primer Mix3 µlTemplate4 µlddH₂O3 µlNote: High ROX model: add 1 µL of 50×High ROX per 50 µL of reaction system; Low ROX model: add 1 µL of 50×High ROX per 500 µL of reaction system.A sufficient amount of reaction system mixture was prepared according to the need, and after the reaction system was prepared and mixed thoroughly, it was added to the reaction wells in a volume of 16 µl per well. Then add the prepared standards and diluted samples into the corresponding reaction wells, the amount of addition is 4µL/well. TE was added to the blank control tube, and the same amount was added at 4 µL/well.It is recommended to use 20 µL for the reaction, if you need to perform a smaller system reaction, reduce the system components in equal proportion.4. qPCR reaction programThe PCR mix of this kit contains a FAM fluorescent probe for the target gene and a VIC fluorescent probe with internal reference to Internal PCR Control (IPC). qPCR program with dual fluorescence of hydrolyzed probes needs to be selected for the assay. Please follow the instructions of the instrument used to set up the qPCR program, and the PCR temperature conditions are as follows:1. Standard curve productionThe standard curve was plotted with reference to the Excel sheet for data processing. The correlation coefficient R2 of the standard curve should be not less than 0.98, and the slope should be located between -3.1 and -3.6 when the Ct value is used as the longitudinal coordinate. If the parameters of the standard curve are unreasonable, it is recommended to repeat the experiment.DNA Standard NameDNA Standard Concentration（ng/µL）DNA Standard 110DNA Standard 22.5DNA Standard 30.625DNA Standard 40.15625DNA Standard 50.03906252. Analysis of results and calculation of concentrationsThe Ct difference between experimental replicate wells for FAM signaling of the target gene should be no more than 0.3, otherwise invalid data need to be deleted or the experiment needs to be repeated, do not use Ct outside the valid Ct range of the standard curve to calculate the concentration of the sample.For specific calculations, please refer to the data processing Excel for this product.If the FAM signal is abnormal, the VIC signal of the internal reference Internal PCR Control (IPC) needs to be analyzed to confirm whether the PCR reaction process is abnormal. If the Ct value of the sample null VIC is significantly larger than that of the standard or blank control wells, it means that the sample inhibits the PCR reaction.matters needing attention1. Before testing, these instructions should be read in detail. It should be operated by personnel with professional experience or qualified by training.2. For use, please mix gently by turning up and down, avoid foaming as much as possible, and use it after centrifugation for a short period of time.3. Avoid repeated freezing and thawing of the product, repeated freezing and thawing may degrade the performance of the product.4. When preparing the reaction solution, please use new or non-contaminated tips and centrifuge tubes to prevent contamination as much as possible... Read More |