| 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 | Inquire | The miRNA extraction kit is specifically designed to isolate and purify miRNAs from various animal tissues, plant tissues, cells, serum, plasma and other samples. It can also extract small molecule RNAs such as siRNA and snRNA that are less than 200 nt, and can also be used for the extraction of The miRNA extraction kit is specifically designed to isolate and purify miRNAs from various animal tissues, plant tissues, cells, serum, plasma and other samples. It can also extract small molecule RNAs such as siRNA and snRNA that are less than 200 nt, and can also be used for the extraction of total RNA. This product combines phenol/guanidine lysis technology and silicon matrix membrane purification technology. The unique lysis solution can effectively inhibit RNases while removing most of DNA and proteins from cell or tissue samples through organic extraction. For some sensitive downstream experiments, if miRNA enrichment is required, this kit can be used to enrich miRNA separately. This product is suitable for a wide range of samples, with high purity of prepared RNA, and can be directly used for sensitive downstream applications, such as Northern Blot analysis, Real Time PCR, Microarray Analysis, etc. M665531Component50 TStorageM665531ATRIzon Reagent60 mL2-8℃. Protect from ligt.M665531BBuffer RWT (concentrate)15 mLRTM665531CBuffer RW2 (concentrate)11 mLRTM665531DRNase-Free Water10 mLRTM665531ESpin Columns RM with Collection Tubes50 setsRTM665531FSpin Columns RS with Collection Tubes50 setsRTM665531GRNase-Free Centrifuge Tubes (1.5 mL)50 EART Self prepared reagents: chloroform, anhydrous ethanol (newly opened or dedicated for RNA extraction).Preparation and important precautions before the experiment:To prevent RNase pollution, attention should be paid to the following aspects:1) Use RNase free plastic products and gun heads to avoid cross contamination.2) Glassware should be dry baked at a high temperature of 180 ℃ for 4 hours before use, while plastic containers can be soaked in 0.5 M NaOH for 10 minutes, thoroughly rinsed with water, and then sterilized under high pressure.3) Prepare the solution using water without RNase.4) Operators should wear disposable masks and gloves, and change gloves frequently during the experiment.2. The extracted samples should avoid repeated freeze-thaw cycles, otherwise it will affect the quantity and quality of miRNA extraction.Before the first use, anhydrous ethanol should be added to Buffer RWT and Buffer RW2 according to the instructions on the reagent bottle label.4. All centrifugation steps should be carried out at room temperature unless otherwise specified, and all operation steps should be carried out quickly.Operation steps:Protocol A: miRNA enrichment (can be directly used for sensitive downstream experiments)1. Sample processing1a Organization: Grind the organization in liquid nitrogen. Add 1 ml of TRIzon Reagent to every 30-50 mg of tissue, shake and mix well. The sample volume shall not exceed one tenth of the volume of TRIzon Reagent.1b Single layer culture of cells: Remove the culture medium, add TRIzon Reagent, and add 1 ml of TRIzon Reagent every 10 cm2 (the amount of lysis solution depends on the area of the culture bottle).1c Cell suspension: Centrifuge to obtain cell precipitate, discard supernatant. Add 1 ml of TRIzon Reagent to every 5 x 106-1 x 107 cells (cells do not require washing).1d Plasma or serum: Take 200 µ Add 5 times the volume of TRIzon Reagent to plasma or serum samples, shake and mix well for 30 seconds.2. After adding TRIzon Reagent to the sample, blow it repeatedly several times to fully crack it. Leave at room temperature for 5 minutes to completely separate the protein nucleic acid complex.3. Optional steps: Centrifuge at 4 ℃ 12000 rpm (~13400 × g) for 5 minutes, take the supernatant, and transfer it to a new centrifuge tube (provided by oneself) (if the sample contains more proteins, fats, polysaccharides, etc., this step can be performed).4. Add chloroform to the supernatant and add 200 to every 1 ml of TRIzon Reagent used µ Chloroform, cover the tube, vigorously shake for 15 seconds, and let it sit at room temperature for 5 minutes.Centrifuge at 5.4 ℃ and 12000 rpm for 15 minutes. The sample is divided into three layers: red organic phase, middle layer, and colorless aqueous phase. Transfer the upper colorless aqueous phase to a new centrifuge tube (self prepared).6. Add 1/3 volume of anhydrous ethanol to the solution obtained in step 5, mix well, and transfer the obtained solution and precipitate together into the adsorption column RM (Spin Columns RM) that has been loaded into the collection tube. If you cannot add all the solution to the adsorption column at once, please transfer it multiple times. Centrifuge at 12000 rpm for 30 seconds, discard the adsorption column RM after centrifugation, and retain the effluent.7. Add 2/3 times the volume of anhydrous ethanol to the solution obtained in step 6 and mix well.8. Transfer the solution and precipitate obtained from the previous step into the adsorption column RS (Spin Columns RS) that has been loaded into the collection tube. If you cannot add all the solution to the adsorption column at once, please transfer it multiple times. Centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column RS back into the collection tube.9. Add 700 to the adsorption column RS µ L Buffer RWT (check if anhydrous ethanol is added before use), centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column RS back into the collection tube.10. Add 500 to the adsorption column RS µ Buffer RW2 (check if anhydrous ethanol is added before use), centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column RS back into the collection tube.11. Repeat step 10.12. Centrifuge at 12000 rpm for 1 minute and discard the waste liquid from the collection tube. Place the adsorption column RS 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 RS, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).13. Place the adsorption column RS in a new RNase free centrifuge tube and add 30-50 to the middle of the adsorption column µ Place RNase Free Water at room temperature for 1 minute, centrifuge at 12000 rpm for 1 minute, collect RNA solution, and store the obtained RNA solution at -70 ℃ to prevent degradation.Attention:1) The volume of RNase Free Water should not be less than 30 µ l. Small volume affects the recovery rate.2) If you want to increase RNA production, you can use 30-50 µ Repeat step 13 for the new RNase Free Water.3) If you want to increase the RNA concentration, you can add the obtained solution back to the adsorption column RS and repeat step 13Protocol B: Extraction of total RNA (including miRNA and other small molecule RNAs<200 nt), steps 1-5 are the same as protocol A.6. Add 1.25 times the volume of anhydrous ethanol to the solution obtained in step 5 and mix well.7. Transfer the solution and precipitate obtained from the previous step into the spin columns RM that have been loaded into the collection tube. If you cannot add all the solution to the adsorption column RM at once, please transfer it multiple times. Centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column RM back into the collection tube.8. Add 700 to the adsorption column RM µ L Buffer RWT (check if anhydrous ethanol is added before use), centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column RM back into the collection tube.9. Add 500 to the adsorption column RM µ Buffer RW2 (check if anhydrous ethanol is added before use), centrifuge at 12000 rpm for 30 seconds, discard the waste liquid in the collection tube, and place the adsorption column RM back into the collection tube.10. Repeat step 9.11. Centrifuge at 12000 rpm for 1 minute and discard the waste liquid from the collection tube. Place the adsorption column RM at room temperature for a few minutes to thoroughly air dry. Attention: The purpose of this step is to remove residual ethanol from the adsorption column RM, which can affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).12. Transfer the adsorption column RM into a new RNase free centrifuge tube and add 30-50 to the middle of the adsorption column µ Place RNase Free Water at room temperature for 1 minute, centrifuge at 12000 rpm for 1 minute, collect RNA solution, and store the obtained RNA solution at -70 ℃ to prevent degradation. Attention: 1) The volume of RNase Free Water should not be less than 30 µ l. Small volume affects the recovery rate.2) If you want to increase RNA production, you can use 30-50 µ Repeat step 12 for the new RNase Free Water.3) If you want to increase the RNA concentration, you can add the obtained solution back to the adsorption column RM and repeat step 12... Read More | DescriptionMetathesis: Ruthenium-Based Metathesis CatalystsRuthenium metathesis catalysts kit I consists of 9 samples of Grubbs 1st and 2nd generation catalysts. These catalysts have applications in ring-closing and ring-opening metathesis, cross-metathesis, ring-opening metathesis polymerization (DescriptionMetathesis: Ruthenium-Based Metathesis CatalystsRuthenium metathesis catalysts kit I consists of 9 samples of Grubbs 1st and 2nd generation catalysts. These catalysts have applications in ring-closing and ring-opening metathesis, cross-metathesis, ring-opening metathesis polymerization (ROMP) and enyne metathesis.Metathesis: Ruthenium-Based Metathesis Catalysts... 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