| Description | The hydroxyl radical (·OH) is the neutral form of the hydroxide ion (OH⁻) and possesses strong oxidizing capacity. Hydroxyl radicals act on biological molecules such as proteins, nucleic acids, and lipids within the body, damaging cellular structure and function, which can lead to The hydroxyl radical (·OH) is the neutral form of the hydroxide ion (OH⁻) and possesses strong oxidizing capacity. Hydroxyl radicals act on biological molecules such as proteins, nucleic acids, and lipids within the body, damaging cellular structure and function, which can lead to metabolic disorders and disease. The hydroxyl radical scavenging capacity is a key indicator of antioxidant ability and is widely used in research on antioxidant health products and pharmaceuticals.Detection Principle: H₂O₂/Fe²⁺ generates hydroxyl radicals via the Fenton reaction. Salicylic acid effectively captures these generated hydroxyl radicals and reacts with them to produce a purple compound, 2,3-dihydroxybenzoic acid. When a substance capable of scavenging hydroxyl radicals is added, it inhibits the formation of this purple product. Therefore, a darker color indicates a higher hydroxyl radical content, and vice versa. The change in absorbance at 520 nm is measured to calculate the sample's hydroxyl radical scavenging capacity.Applicable Samples: Animal and plant tissues, serum (plasma), cells, bacteria, cell culture supernatants, fruit juice, honey, urine, and other samples.P1501782Component48 T96 TStorageP1501782AFerrous Salt10 mL20 mL2-8℃. Store in the dark.P1501782BH₂O₂5 mL10 mL2-8℃. Store in the dark.P1501782CSalicylic Acid10 mL20 mL2-8℃. Store in the dark.Please check the quantities of all components before starting the experiment.An additional 10% of each component is provided beyond the specified volumes for standard curve preparation or preliminary experiments.User-Prepared Instruments and ReagentsTypeNameNotesInstrumentMicroplate ReaderCapable of measuring absorbance at 520 nm.Consumables96-well MicroplateStandard microplate.ReagentsPBS (pH7.4)For washing samples.OthersHomogenizer (for tissue samples), Incubator, Ice Box, Refrigerated Centrifuge, Adjustable Micropipettes and TipsUsing a multi-channel pipette can improve efficiency for large-scale assays.Experimental Procedure1. Reagent PreparationReagent NameReagent PreparationNotesFerrous SaltReady-to-use; Equilibrate to room temperature before use.Store at 4°C protected from light. Corrosive. Use appropriate personal protective equipment.H₂O₂Ready-to-use; Equilibrate to room temperature before use.Store at 4°C protected from light.Salicylic AcidReady-to-use; Equilibrate to room temperature before use.Store at 4°C protected from light. Irritating to skin and mucous membranes. Use appropriate personal protective equipment.2. Sample PreparationNote: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for one month. To compare the hydroxyl radical scavenging capacity of different samples, the dilution factor must be the same for the same batch of samples, and extracts or drugs should be prepared at the same concentration.2.1 Animal Tissue SamplesWeigh approximately 0.1 g of tissue, add 1 mL of deionized water, and homogenize in an ice bath. Centrifuge at 10,000 g, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.2.2 Plant Tissue SamplesWeigh approximately 0.1 g of tissue, add 1 mL of deionized water and grind. Sonicate in an ice bath for 5 minutes (power 20% or 200W, pulse 3s on, 7s off, repeat 30 times). Centrifuge at 10,000 g, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.2.3 Cells or BacteriaCollect 5×10⁶ cells or bacteria into a centrifuge tube. Wash with pre-cooled PBS, centrifuge, and discard the supernatant. Add 1 mL of deionized water. Sonicate in an ice bath for 5 minutes (power 20% or 200W, pulse 3s on, 7s off, repeat 30 times). Centrifuge at 10,000 g, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.2.4 Serum (Plasma) and Other Protein-Rich or Turbid LiquidsTake 0.1 mL of sample, add 1 mL of deionized water and mix well. Centrifuge at 10,000 g, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.2.5 Honey, Urine, and Other Clear Liquids with Low Protein ContentAssay directly.2.6 Extracts or DrugsCan be prepared to a specific concentration, e.g., 0.5 mg/mL.3. Assay Steps3.1 Microplate Reader Preparation: Preheat for at least 30 minutes. Set the wavelength to 520 nm.3.2 Assay System Setup: Perform the following operations in a 96-well plate. The Blank and Standard wells only need to be set up 1-2 times. Each test well requires a corresponding control well.ReagentBlank Well (µL)Standard Well (µL)Test Well (µL)Control Well (µL)Ferrous Salt40404040H₂O₂040400Deionized Water120804080Salicylic Acid40404040Sample0040403.3 Absorbance Measurement: Mix well, incubate at 37°C for 20 minutes. Read the absorbance at 520 nm, recorded as A blank, A standard, A test, and A control respectively.4. Calculation of ResultsBoth the derived formula and the simplified formula provided below are equivalent.4.1 Data ProcessingCalculate ΔA test = A test - A control Calculate ΔA standard = A standard - A blank 4.2 Calculation of Hydroxyl Radical Scavenging RateHydroxyl Radical Scavenging Rate D% = (ΔA standard - ΔA test ) / ΔA standard × 100%5. Representative ResultsExample: 0.1 g of nectarine pulp was taken and assayed according to the procedure using a 96-well plate.Measured: ΔA standard = A standard - A blank = 1.020 - 0.051 = 0.969ΔA test = A test - A control = 0.465 - 0.052 = 0.413Calculated Hydroxyl Radical Scavenging Rate D% = (0.969 - 0.413) / 0.969 × 100% = 57.38%Precautions1. Before formal testing, it is recommended to perform a preliminary test with 2-3 samples expected to have significant differences.2. For tissue samples, cell samples, etc., results can be normalized between samples by measuring protein concentration. Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) is recommended.3. This kit is compatible with spectrophotometer detection. Adjust the reagent preparation volumes proportionally according to the spectrophotometer's requirements.4. Biochemical reagents are generally irritating and potentially biologically toxic. For your safety and health, implement appropriate biosafety precautions throughout the experiment, including wearing lab coats, masks, gloves, and head covers. Perform experiments in a fume hood or biosafety cabinet.5. This product is for research use only. Not for use in clinical diagnosis.Frequently Asked QuestionsQ: What should I do if the measured ΔA test for the sample is too high or too low?A: If ΔA test < 0.02, appropriately increase the sample volume and re-run the assay. If ΔA test > ΔA standard, further dilute the sample with deionized water or reduce the amount of sample used for extraction, and re-run the assay... 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 | D665729 Component 50 T Storage D665729A Conversion Buffer CR 5×1 mL RT D665729B Buffer CL 30 mL RT D665729C Buffer MD 0.4 mL RT D665729D Buffer DB 10 mL RT D665729E Buffer WB (concentrate) 10 mL RT D665729F Buffer GW1 (concentrate) 13 mL RT D665729G Buffer GW2 (concentrate) 15 mL RT D665729H D665729 Component 50 T Storage D665729A Conversion Buffer CR 5×1 mL RT D665729B Buffer CL 30 mL RT D665729C Buffer MD 0.4 mL RT D665729D Buffer DB 10 mL RT D665729E Buffer WB (concentrate) 10 mL RT D665729F Buffer GW1 (concentrate) 13 mL RT D665729G Buffer GW2 (concentrate) 15 mL RT D665729H Buffer EB 4 mL RT D665729I Buffer PS 10 mL RT D665729J Spin Columns DF 50 Pcs 2-8 ℃ D665729K Collection Tubes 50 Pcs RTProduct Introduction:The basic principle of this reagent kit is that after DNA is treated with sodium bisulfite, unmethylated cytosine can be transformed into uracil, while methylated cytosine remains unchanged. And adopting an innovative high-temperature treatment method, the transformation time is greatly shortened, the transformation efficiency is improved, and the transformation efficiency can reach over 99%. At the same time, using a silicon-based membrane purification column, DNA can be recovered and purified from the methylated solution through a simple binding washing elution step. The recovered DNA has high purity and good integrity, and can be directly used for sequencing, methylated PCR detection, chip analysis, connection and transformation, enzyme digestion, labeling, microinjection, PCR and in vitro transcription and other molecular biology experiments.Self prepared reagents: anhydrous ethanol, 75% ethanol.Preparation and important precautions before the experiment1. Product usage method:(1) 10 times packaging preparation method: CT Conversion Agent is a solid mixture that must be prepared before first use. Add 2 ml sterile water and 100 µ M-Dissolving Buffer and 300 µ Add M-Diffusion Buffer to the CT Conversion Agent tube. Dissolve at 55 ° C and shake until completely dissolved. Store the CT Conversion Agent solution at room temperature (20 ° C-30 ° C) in the dark before use. The CT Conversion Agent for each tube is designed for 10 DNA treatments. In order to achieve better results, the prepared CT Conversion Agent should be used immediately. If not used immediately, the CT Conversion Agent solution can be stored at -20 ° C for 1 week. Before use, be sure to thaw the stored CT Conversion Agent solution at room temperature and mix thoroughly by shaking or inverting for 2 minutes, CT Conversion Reagent is sensitive to light, so it is important to minimize exposure to light as much as possible.(2) 50 times packaging preparation method: CT Conversion Agent and M-Dissolving Buffer are solid mixtures that must be prepared before first use. Add 5 ml of sterile water to the M-Dissolving Buffer and shake to dissolve. After all the solids have dissolved, transfer all the solution from the M-Dissolving Buffer tube to the CT Conversion Agent tube and add 5.5 ml of sterile water. Add 1.5 ml of M-Dilution Buffer to the CT Conversion Agent tube. Dissolve at 55 ° C and shake until completely dissolved. Store the CT Conversion Agent solution at room temperature (20 ° C-30 ° C) in the dark before use. The CT Conversion Agent for each tube is designed for 50 DNA treatments. In order to achieve better results, the CT Conversion Agent should be used immediately after preparation. If not immediately used, the CT Conversion Agent solution can be stored at -20 ° C for 1 week. Before use, be sure to thaw the stored CT Conversion Agent solution at room temperature and mix thoroughly by shaking or inverting for 2 minutes, CT Conversion Reagent is sensitive to light, so it is important to minimize exposure to light as much as possible.2. Before the first use, anhydrous ethanol should be added to the M-Wash Buffer according to the instructions on the reagent bottle label.Operation stepsThe range of DNA prepared each time is 1 ng-4 µ Between g, the optimal amount is 500 ng-2 µ G.1. Take 20 µ Add DNA sample into centrifuge tube (self provided), and if the sample amount is insufficient, replenish with water up to 20 µ L.2. Add 2.2 to the DNA sample µ Mix the sample well with the M-Dilution Buffer of l.3.42 ℃ water bath for 30 minutes.4. Add 220 to the sample obtained from the previous step µ Prepare the CT Conversion Agent solution, mix well, and incubate in an 80 ℃ constant temperature water bath in a dark place for 60 minutes.5. Add 480 to the solution in the previous step µ M - Buffer PA, gently mix upside down.6. Column balance: Add 200 to the spin columns DS that have been loaded into the collection tube µ Centrifuge at 12000 rpm (~13400 × g) for 2 minutes, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.7.Add all the solution obtained from step 5 to the adsorption column (already loaded into the collection tube), let it stand at room temperature for 2 minutes, 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.Attention: The maximum capacity of the adsorption column is 750 µ l. If the sample volume is greater than 750 µ L can be added in batches.8. Add 500 to the adsorption column µ Centrifuge at 12000 rpm for 1 minute using M-Buffer PA, discard the waste liquid from the collection tube, and place the adsorption column in the recovery tube.9. Add 650 to the adsorption column µ M-Wash Buffer (please check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column in the collection tube.10.12000 rpm for 2 minutes, discard the waste liquid, and 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 will affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).11. Place the adsorption column into a new centrifuge tube (provided by oneself), and add 20 drops to the middle position of the adsorption membrane in the air µ M-Elution Buffer (pH 8.5), leave at room temperature for 2 minutes. Collect DNA solution by centrifugation at 12000 rpm for 1 minute.12. Collect 20 µ Add 2.2 to DNA µ M-Diffusion Buffer, let it stand at room temperature for 30 minutes.13. Add 500 to the solution µ After pre cooling anhydrous ethanol, invert and mix well, and place the solution at -20 ℃ to precipitate for 30 minutes (overnight precipitation is more effective).14.12000 rpm for 15 minutes and gently discard the supernatant.15. Add 75% ethanol, centrifuge at 12000 rpm for 1 minute, pour out the supernatant, wait for ethanol to evaporate at room temperature, then add 20 µ Dissolve the M-Elution buffer and store the DNA at -20 ℃. The DNA collected in this step can be used for subsequent related experiments... Read More | Inquire | 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 |