| 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 | Alanine Aminotransferase (ALT), also known as serum glutamic-pyruvic transaminase (SGPT), is a pyridoxal-phosphate-dependent enzyme that catalyzes the reversible transfer of an amino group from alanine to α-ketoglutarate, generating pyruvate and glutamate. ALT is found primarily in liver and Alanine Aminotransferase (ALT), also known as serum glutamic-pyruvic transaminase (SGPT), is a pyridoxal-phosphate-dependent enzyme that catalyzes the reversible transfer of an amino group from alanine to α-ketoglutarate, generating pyruvate and glutamate. ALT is found primarily in liver and serum, but occurs in other tissues as well. Hepatocellular injury often results in an increase of serum ALT levels and serum ALT levels can be used as a marker for liver injury.ALT Activity Assay kit has been used to determine the activity of alanine aminotransferase (ALT) in serum samples... Read More | Product contentG665801Component100 TStorageG665801A2×GoldStar Probe One Step Buffer1.4 mL-20℃. Avoid freeze/ Thaw cycle. Protect from light.G665801BGoldStar Probe One Step EnzymeMix100 µL-20℃. Avoid freeze/ Thaw cycle. Protect from light.G665801C50×High ROX50 µL-20&#Product contentG665801Component100 TStorageG665801A2×GoldStar Probe One Step Buffer1.4 mL-20℃. Avoid freeze/ Thaw cycle. Protect from light.G665801BGoldStar Probe One Step EnzymeMix100 µL-20℃. Avoid freeze/ Thaw cycle. Protect from light.G665801C50×High ROX50 µL-20℃. Avoid freeze/ Thaw cycle. Protect from light.G665801DRNase-Free Water1.5 mL-20℃. Avoid freeze/ Thaw cycle. Product Introduction This product is a specialized kit for one-step Real-Time RT-qPCR using the probe method (TaqMan, Molecular Beacon, etc.). When using this product for Real Time RT-qPCR reaction, reverse transcription and quantitative PCR are carried out in the same reaction system, and there is no need to add reagents or open the cap of the tube during the reaction process, which avoids contamination and improves the experimental efficiency at the same time. With high detection sensitivity, strong fluorescence signal and high signal-to-noise ratio, this product is very suitable for the detection of RNA viruses and other trace RNA. The special buffer system contained in this product can maximize the effectiveness of reverse transcriptase and DNA polymerase at the same time and improve the efficiency of the reaction. A wider linear range can be obtained with this product, more accurate quantification of the target gene, good reproducibility and high confidence.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 (G665836) Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96 and others. Instruments that require High ROX calibration (G665801) ABI Prism 7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, and others.matters needing attention1.Before using the reagents in this kit, please mix them gently by turning them up and down to avoid foaming as much as possible, and use them after brief centrifugation.2.This product uses RNA as the template for one-step RT-PCR experiment, RNase contamination should be avoided during operation, it is recommended to operate RNA in a special area, use special instruments and consumables, the operator with a mask and disposable gloves and often change the gloves, the experiment-related consumables should be processed with 0.1% DEPC (diethyl ether pyrocarbonate) aqueous solution for 12 hours at 37℃, and autoclaved for 30 minutes before use. The consumables should be treated with 0.1% DEPC (diethylpyrocarbonate) aqueous solution at 37℃ for 12 hours and autoclaved for 30 minutes.3.Repeated freezing and thawing of each reagent in this kit should be avoided as much as possible; repeated freezing and thawing may degrade the product performance.4.This kit must use specific primers, the choice of primers can be selected according to specific experiments, the good or bad primer design directly affects the results of RT-qPCR reaction, the design of primers need to consider the GC content, primer length, primer position, the secondary structure of the PCR product and other factors, it is recommended to use a professional primer design software for design.5.This kit is recommended to use specific probes, and it is recommended to use professional design software for designing.UsageThe following examples are conventional reaction systems and conditions, which should be improved and optimized according to the different templates, primer structures and target fragment sizes in actual operation. (Please prepare the reaction solution on ice.)1. Dissolve RNA template, primers, 2× GoldStar Probe One Step Buffer, GoldStar Probe One Step EnzymeMix and RNase-Free Water and set aside on ice.2. PCR reaction system:reagents25µl reaction systemfinal concentration2×GoldStar Probe One Step Buffer12.5µl1×Forward Primer, 10µM0.5µl0.2µM¹⁾Reverse Primer, 10µM0.5µl0.2µM¹⁾Probe, 10µM0.5µl0.2µM²⁾GoldStar Probe One Step EnzymeMix1.0µl RNA TemplateXµl10pg-100ng³⁾50 x Low ROX or High ROX (optional)⁴⁾0.5µl1×RNase-Free WaterUp to 25µlNote: 1) Usually, better results can be obtained with a primer concentration of 0.2 µM, and 0.1-1.0 µM can be used as a reference for setting the range.(2) The concentration of the probe used is related to the fluorescence quantitative PCR instrument used, the type of probe, and the type of fluorescent labeling substance, please refer to the instrument manual or the specific requirements for the use of each fluorescent probe for the adjustment of the concentration in actual use.(3) Usually the amount of RNA template is 10pg-100ng as a reference. Since the templates of different species contain different copy numbers of target genes, the templates can be diluted in gradient to determine the optimal amount of template to use.(4) The excitation optical system varies from instrument to instrument, choose to add 50×Low ROX or 50×High ROX according to the instrument using fluorescence quantification.3. Mix well, centrifuge briefly, and collect the solution at the bottom of the tube.4.RT-PCR reaction conditions:Note: 1) The hot start enzyme used in this product must be activated under the condition of pre-denaturation 95℃, 5-10min. 2) It is recommended to use the two-step PCR reaction program, if you can not get good experimental results due to the use of primers with lower Tm value, etc., you can try to carry out the three-step PCR amplification, and the annealing temperature should be set in the range of 56℃-64℃ as a reference... Read More | Inquire | 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 |