| 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 | Product content:ComponentG665836100 rxnsG665836100 rxnsG665836100 rxns2×GoldStar Probe One Step Buffer1.4 ml1.4 ml1.4 mlGoldStar Probe One Step EnzymeMix100 µl100 µl100 µl50×Low ROX-50 µl-50×High ROX--50 µlRNase-Free Water1.5 ml1.5 ml1.5 mlProduct IntroductionProduct content:ComponentG665836100 rxnsG665836100 rxnsG665836100 rxns2×GoldStar Probe One Step Buffer1.4 ml1.4 ml1.4 mlGoldStar Probe One Step EnzymeMix100 µl100 µl100 µl50×Low ROX-50 µl-50×High ROX--50 µlRNase-Free Water1.5 ml1.5 ml1.5 mlProduct Introduction：This product is a specialized reagent kit for one-step Real Time RTqPCR using probe methods (TaqMan, Molecular Beacon, etc.). When using this product for Real Time RT qPCR reaction, reverse transcription and quantitative PCR are requiredConducted in the same reaction system, there is no need to add reagents or open the tube cap during the reaction process, avoiding contaminationThis has improved the efficiency of the experiment. This product has high detection sensitivity, strong fluorescence signal, and high signal-to-noise ratio, making it very suitable forDetection of RNA viruses and other trace amounts of RNA. The special buffering system it contains can enable reverse transcriptase to interact with DNA polymeraseMaximize the effectiveness and improve reaction efficiency. By using this product, a wider linear range can be obtained, which is beneficial for the target base Due to more accurate quantification, good repeatability, and high reliability.ROX dye is used to correct the fluorescence signal error generated between wells in quantitative PCR instruments, and is generally used for ABIReal Time PCR amplification equipment from companies such as Stratagene. The excitation optical systems of different instruments vary, thereforeThe concentration of ROX dye must be matched with the corresponding fluorescence quantitative PCR instrument.matters needing attention：1. Before using the reagents in this reagent kit, please gently mix them upside down to avoid foaming as much as possible, and use them after brief centrifugation. 2. This product uses RNA as a template for one-step RT-PCR experiments, and RNase contamination should be avoided during the operation process,2.It is recommended to perform RNA operations in a dedicated area, using specialized instruments and consumables. Operators should wear masks and disposable gloves and frequently change gloves. Experimental consumables should be treated with a 0.1% DEPC (diethyl pyrocarbonate) aqueous solution at 37 ℃ for 12 hours and sterilized under high pressure for 30 minutes before use.3. Each reagent in this kit should avoid repeated freezing and thawing as much as possible, as repeated freezing and thawing may lead to a decrease in product performance.4. This reagent kit must use specific primers, and the selection of primers can be based on specific experiments. The quality of primer design directly affects the results of RT qPCR reaction. When designing primers, GC content, primer length, and primer should be considered Due to factors such as location, secondary structure of PCR products, it is recommended to use professional primer design software for design.5. It is recommended to use specific probes in this reagent kit and use professional design software for design. Usage: The following examples are typical reaction systems and conditions. In practical operation, corresponding improvements and optimizations should be made based on the differences in template, primer structure, and target fragment size. (Please prepare the reaction solution on ice)1. Dissolve the RNA template, primers, 2xGoldStar Probe One Step Buffer, GoldStar Probe One Step EnzymeMix, and RNase Free Water and place them on ice for later use.2. PCR reaction system: reagent 25 µl Reaction system final concentration 2×GoldStar Probe One Step Buffer 12.5 µl 1× Forward Primer，10 µM 0.5 µl 0.2 µM 1） Reverse Primer，10 µM 0.5 µl 0.2 µM 1） Probe ，10 µM 0.5 µl 0.2 µM 2） GoldStar Probe One Step EnzymeMix 1.0 µl / RNA Template X µl 10 pg – 100 ng3） 50×Low ROX or High ROX （optional）4） 0.5 µl 1× RNase-Free Water up to 25 µl /Note: 1) Typically, the primer concentration is 0.2 µ M can achieve good results, ranging from 0.1 to 1.0 µ M serves as a reference for setting the range. 2) The concentration of the probe used is related to the fluorescent quantitative PCR instrument used, the type of probe, and the type of fluorescent labeling substance. Please refer to the instrument manual or the specific usage requirements of each fluorescent probe for concentration adjustment during actual use.3) The amount of RNA templates is usually based on 10 pg-100 ng as a reference. Due to the different copy numbers of target genes contained in templates of different species, gradient dilution can be applied to the templates to determine the optimal template usage.4) The excitation optical systems of different instruments vary, and depending on the instrument used for fluorescence quantification, 50 x Low ROX or 50 x High ROX can be added.3. Mix well, centrifuge briefly, and collect the solution to the bottom of the tube.4. RT-PCR reaction conditions steps temperature time / Reverse Transcription 45℃ 10 min / PCR pre denaturation 95℃ 10 min / denaturation 95℃ 15s 30-40cycle Annealing/Extension 60℃ 45s 30-40cycleAttention:1) The hot start enzyme used in this product must be activated under pre denaturation conditions of 95 ℃ and 5-10 minutes.2) It is recommended to use a two-step PCR reaction program. If good experimental results cannot be obtained due to the use of primers with lower Tm values, a three-step PCR amplification can be attempted. The annealing temperature should be set within the range of 56 ℃ -64 ℃ as a reference... Read More | Inquire | DescriptionPhoto KitAlysis Starter Kit enables screening of 24 micro-scale simultaneous photocatalytic reactions with consistent and reproducible photon intensity. User guide is provided in the below hyperlink.Photo KitAlysis Operating InstructionsComponents:Photo KitAlysis LED ControllerBlue LED DescriptionPhoto KitAlysis Starter Kit enables screening of 24 micro-scale simultaneous photocatalytic reactions with consistent and reproducible photon intensity. User guide is provided in the below hyperlink.Photo KitAlysis Operating InstructionsComponents:Photo KitAlysis LED ControllerBlue LED Array (470 nm)Photo KitAlysis Reaction BlockTorque screwdriverSmall screwdriver to easily remove torqued screws after reaction is completeFeatures:Designed and tested by synthetic chemists.Controller provides repeatable milliamp selection for photon intensity0-30 mA variable LED output3 different LED options: blue (470 nm, included), green (527 nm, sold separately), and white (sold separately)Non-magnetic LED baseChemically resistant LED coverPTFE coated cablingDesigned to be used withPhoto KitAlysis High-Throughput Reaction Screening Kit(sold separately).Best when used withKitAlysis Benchtop Inertion Box(sold separately)... Read More | Product introduction：PMA qPCR live bacteria detection kit provides an effective means to detect bacterial activity. This kit provides a mixture of PMA dye and SYBR green dye based qPCR. The optimal amount of dye and the number of samples that can be processed may vary depending on the type ofProduct introduction：PMA qPCR live bacteria detection kit provides an effective means to detect bacterial activity. This kit provides a mixture of PMA dye and SYBR green dye based qPCR. The optimal amount of dye and the number of samples that can be processed may vary depending on the type of sample. PMA is a DNA binding dye with high affinity, especially with double stranded DNA. The dye itself has weak fluorescence, but it can emit brighter fluorescence after binding with nucleic acids. PMA is impermeable to the cell membrane, so it can selectively modify the DNA of dead cells with damaged membrane. After bllight (~464 nm) photolysis of PMA modified DNA, the photoreactive azido group on PMA is converted into highly reactive azene radical, which reacts with any hydrocarbon moiety near the DNA binding site to form a stable covalent nitrogen carbon bond, resulting in permanent DNA modification. This modification process will make the DNA insoluble, and it will be lost together with cell debris in the later genomic DNA extraction process. The unbound PMA remaining in the solution reacts with water molecules under strong light irradiation and decomposes into hydroxylamine compounds without cross-linking activity, so that it can no longer covalently bind DNA. Based on this characteristic of PMA, our company combines PMA and qPCR technology to form a new detection method - PMA qPCR, which is used for the screening of live bacteria. At present, the method has been validated in a variety of bacterial strains as well as yeast, fungi, viruses and parasites. The treatment of complex samples, such as feces or soil, may require optimization of sample dilution, dye concentration, and light treatment time. Treatment of diluted samples, such as water testing, may require filtration or concentration prior to dye treatment. Component： Instruction： Precautions before use：1.This live bacteria detection kit distinguishes dead bacteria and live bacteria according to cell membrane permeability. Many methods of killing bacteria cause damage to the cell membrane and are therefore compatible with this kit. But some methods, such as ultraviolet irradiation, may not immediately cause cell membrane rupture. Therefore, before selecting this kit, it is necessary to carry out literature search and pre-experiment to determine whether the kit is suitable for the bacterial type and killing method you choose. 2.After PMA treatment, the bacteria need to be photolyzed to covalently bind the dye to dead cell DNA. Photolysis operations can use blue or white light sources. Generally speaking, the brighter the lamp, the higher the efficiency of the photolysis step. Non-LED lamps ( such as halogen lamps ) may heat your sample and have a negative impact on the analysis. Ice is required to cool the sample during irradiation. 3.Sample can be cryopreservation after photolysis. Frozen samples before PMA treatment photolysis may damage the cell membrane and produce false negative results. If the sample needs to be frozen before detection, it is recommended to perform a pre-experiment first. 4.Part of the mechanism of PMA is to remove PMA covalently modified DNA from the sample by precipitation ; therefore, when extracting genomic DNA, it is necessary to use the same volume of genomic DNA eluent for volume normalization. The positive control can use the genomic DNA of living cells. 5.In order to verify the effectiveness of PMA in the test sample, the Ct ( dCt ) changes between- / + PMA can be compared. Experimental materials ( self-provided )：①Light source ( used for the photolysis step after PMA modified DNA ) ;② Bacterial genomic DNA extraction kit ; Experimental procedure： 1.Suck 10 µL of E.coli bacterial solution in liquid LB medium, and culture E.coli in the bacterial incubator overnight or longer to the logarithmic growth phase ( OD600 ≈ 1.0 ) ; Note : The culture time is adjusted according to the experiment. 2.Two portions of live E.coli, 400 µL each, were placed in a clean centrifuge tube ; 3. ( Recommended ) Preparation of dead E.coli. If the dead E.coli is needed as a control, the dead E.coli can be obtained by heating the living E.coli in a water bath at 95 °C for 5 min, or at 58 °C for 3 h. the subsequent operation of the dead E. coli is the same as that of the living E. coli ; 4.Two copies of live E.coli, one without PMA treatment, and one with 25 µM PMA treatment ( the optimal PMA concentration for treating different types or different sources of bacteria needs to be consulted in the relevant literature ) ; 5.The PMA-treated samples were placed on a shaker at room temperature and incubated in the dark for 10 min to fully mix the dye with the sample ; 6.Exposure of the sample, you can use blue or white light source, irradiation time to explore their own. For example, a 60 W blue light can be used for 15 min. Note : 1 If a halogen lamp is used, we recommend that the PMA-treated sample tube be placed on an ice block 20 cm away from the light source. Ice should be placed in a transparent tray. Adjust the light source to point directly to the sample, photolysis for 5-15 min ; if the bacteria obtained from the environment are directly used for experiments, due to the complexity or turbidity of the environmental samples, the photolysis time needs to be prolonged appropriately. 7.Treated and untreated live E.coli 5000 × g, centrifuged for 10 min, remove the supernatant ; 8.Select the appropriate genomic DNA extraction kit according to the sample type, and use the same elution volume for each group of samples when elution DNA. Note : DNA extraction steps refer to the instructions of the kit used. Part of the mechanism of action of PMA is to remove PMA-bound DNA from the sample by precipitation ; therefore, when extracting genomic DNA, each group should use the same volume of genomic DNA eluent for volume normalization ( the amount of genomic DNA extracted from dead bacteria and live bacteria is inconsistent, so the concentration of the two is significantly different ). 9.Preparation of reaction mixture according to the following system : Note : 1 For the DNA extracted by commercial DNA extraction kit, the qPCR template was optimized with 2 µL as the initial volume ; 2 The template volume should not exceed 10 % of the final reaction volume ; 3 Template concentration : gDNA as template, usually 1-10 ng ; the final concentration of PCR primers is usually 0.4µM, which can get better results. When the reaction performance is poor, the primer concentration can be adjusted in the range of 0.2-1µM. 10.Slightly vortex the reaction mixture, transfer the fixed volume to the PCR tube. 11. Test procedureNote : 1 The extension time is adjusted according to the instrument ; the Taq enzyme in mix can be activated within 2 min, but the genomic DNA may require longer denaturation time, which can be increased at this time, and the specific denaturation time can be adjusted according to the sample type. 12. ( Optional ) Data analysis Using live bacteria and dead bacteria as controls, the number of live cells in the sample was analyzed and calculated. It is recommended to verify the suitability of primers and PCR procedures before starting PMA qPCR detection of live bacteria. Calculation of dead and living bacteria control dCt ( 1 ) After the end of qPCR, the Ct value of each sample was calculated by instrument software ; ( 2 ) By calculating the dCt of each control bacteria, it was judged whether PMA successfully inhibited the amplification of dead bacterial DNA. The calculation is as follows : dCt live = Ct ( live, PMA treated ) -Ct ( live, PMA untreated ) dCt die = Ct ( die, PMA treated ) -Ct ( die, PMA untreated ) ( 3 ) The dCt expectation of living bacteria is close to 0 ± 1, which indicates that PMA does not affect the amplification of living cell DNA ; ( 4 ) The expected value of dCt of dead bacteria is greater than 4 ( dCt is 4 means that it is reduced by about 16 times, that is, 94 % of dead bacterial DNA is removed ; a dCt of 8 indicated a decrease of about 250 times, that is, 99.6 % of the dead bacterial DNA was removed ).( 5 ) The dCt of dead bacteria depends on many factors, including : strain / cell type ; the way bacteria are killed ; the concentration of PMA used ; amplified sequence length. 3. Calculation of the proportion of viable ( optional ) bacteria If the control results of dead and live bacteria are normal, the proportion of live bacteria in the sample can be calculated.( 1 ) Calculate the dCt value of the sample : dCt sample = Ct ( sample, PMA treated ) -Ct ( sample, PMA untreated ) ( 2 ) Conversion of dCt value to live bacteria ratio : PMA inhibition multiple = 2 ( sample dCt ) Viable bacteria % = 100 / PMA inhibition multiple 14. ( Optional ) Calculate the absolute number of live bacteria If you want to calculate the absolute number of viable bacteria in the sample, you need to use a known number of target bacteria genomic DNA to make a standard curve. It is recommended that the diluted concentrations of several groups of genomes are within the range of the qPCR analysis system. ( 1 ) qPCR was performed with the appropriate genome, and the Ct value was used as the ordinate, and the number of cells was used as the abscissa. The R2 value is calculated to determine the linearity, and the slope and y-axis intercept are displayed.( 2 ) Calculate the copy number of the experimental samples : Ct = slope * cell number + y axis intercept ( y = mx + b ) Bacterial count sample = ( Ct-y axis intercept ) / slope Note : The live bacterial DNA was not lost during the purification process. Examples : Product parameters：Pma: ex = 464 nm; Ex/em = 510/610 nm (following photolysis and reaction with dna/rna)Scope of application：Live bacteria detection Matters needing attention：1.Please instantaneously centrifuge the product to the bottom of the tube before use, and then carry out subsequent experiments ; 2.the kit components contain fluorescent dyes, and attention should be paid to avoiding light during use and preservation ; 3.For your safety and health, please wear experimental clothes and disposable gloves... Read More |