| Description | SpeciesGene IDAccessionECSourceLengthMWTag2019-nCoV43740578YP_009725301.13.4.22.69E. coli306aa~33.8kDa-About this proteinName2019-nCoV Main ProteaseSynonyms2019-nCoV Main Protease (Mpro)/ 3C-like Protease (3CLpro), Mpro/3CLpro, 2019-nCoV 3C-like Proteinase, main proteinase, 3C-like proteinase (NSP5)SpeciesGene IDAccessionECSourceLengthMWTag2019-nCoV43740578YP_009725301.13.4.22.69E. coli306aa~33.8kDa-About this proteinName2019-nCoV Main ProteaseSynonyms2019-nCoV Main Protease (Mpro)/ 3C-like Protease (3CLpro), Mpro/3CLpro, 2019-nCoV 3C-like Proteinase, main proteinase, 3C-like proteinase (NSP5), Coronavirus endopeptidase C30, chymotrypsin-like protease, coronavirus 3C-like protease, coronavirus M(pro), coronavirus main proteaseBackground2019-nCoV is a positive-sense single-stranded RNA virus and has high homology with SARS-CoV and MERS-CoV. After infecting host cells, the coronaviral ORF1a/b RNA is first translated with the help of host cells into two polyprotein precursors (PP1a and PP1ab) which are then cleaved by Mpro and papain like proteases to produce multiple nonstructural proteins. These nonstructural proteins are involved in the production of viral subgene RNA and four structural proteins (envelope/E protein, membrane/M protein, spike/S protein and nucleocapsid/N protein), which in turn complete the reproduction and release of the progeny virus. Since the Mpro protease plays a vital role in the life cycle of the virus and there is no homologous protein in the human body, the Mpro main protease is an ideal target for developing antiviral drugs. Meanwhile because Mpro is highly conservative among beta coronaviruses, the Mpro inhibitors selected have a broad-spectrum anti-coronavirus ability and may even be used in curing other animal diseases such as porcine coronavirus.Aladdin's 2019-nCoV Main Protease is purified using the PerfectProtein™ Platform developed by aladdin. It is recombinantly expressed in E. coli without any additional tags or amino acids, ensuring complete identity with the natural Main Protease in 2019-nCoV virus.s2019-nCoV inhibitor screening; enzyme activity or structural studiesPhysical AppearanceliquidBiological Activity≥300U/g or 300µmol/min/gUnit DefinitionOne unit is defined as the amount of enzyme that catalyzes the production of 1µmol of MCA-AVLQ per minute at 37℃ under pH7.0. Concentration~1mg/mlPurity≥ 95% by SDS-PAGE.Formulation500mM Tris, 150mM NaCl, 1mM EDTA, 50% Glycerol, pH7.3Recommended UsageFor inhibitor screening, add 0.2-1µg of this product generally in a reaction volume of 200µl.Amino Acid SequenceSGFRKMAFPS GKVEGCMVQV TCGTTTLNGL WLDDVVYCPR HVICTSEDML NPNYEDLLIR KSNHNFLVQA GNVQLRVIGH SMQNCVLKLK VDTANPKTPK YKFVRIQPGQ TFSVLACYNG SPSGVYQCAM RPNFTIKGSF LNGSCGSVGF NIDYDCVSFC YMHHMELPTG VHAGTDLEGN FYGPFVDRQT AQAAGTDTTI TVNVLAWLYA AVINGDRWFL NRFTTTLNDF NLVAMKYNYE PLTQDHVDIL GPLSAQTGIA VLDMCASLKE LLQNGMNGRT ILGSALLEDE FTPFDVVRQC SGVTFQNotePrecautions：Before opening the tube, centrifuge briefly to collect liquid at the bottom of the tube.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.Instructions for Use：1. For the usage of this product, please refer to the manual of ’s Coronavirus Mpro/3CLpro Activity Fluorometric Assay Kit or other relevant literature, depending on the experimental purposes.2. Please refer to Figure 1 for the performance of this product in cleaving the CoV Main Protease Fluorogenic Substrate, MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2, into a fluorescent product with Ex/Em of 325/393nm.Figure 1. Digestion of MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2 by different amounts of 2019-nCoV Main Protease (Mpro/3CLpro) (, P2320). This figure is for reference only, which may vary due to different experimental conditions... Read More | Inquire | This product is a mixture of fast reverse transcription reagents. The 5 x EasyQuick RT MasterMix contains all the reagents required for reverse transcription from RNA templates to cDNA first strand, including EasyQuick RT Reversase, RNase Inhibitor, Random 6 mers, Oligo dT Primer, dNTP, EQ-RT BufferThis product is a mixture of fast reverse transcription reagents. The 5 x EasyQuick RT MasterMix contains all the reagents required for reverse transcription from RNA templates to cDNA first strand, including EasyQuick RT Reversase, RNase Inhibitor, Random 6 mers, Oligo dT Primer, dNTP, EQ-RT Buffer, etc. The reverse transcription efficiency of this product is high, and it can perform a good reverse transcription reaction on a small amount of RNA templates. The fluorescence quantitative template cDNA first strand synthesis can be completed in 15 minutes. This reagent kit is very convenient and fast to operate, and only RNA templates and water need to be added for reverse transcription reaction, making it particularly suitable for high-throughput detection.E665905Component200 TStorageE665905A5×EasyQuick RT MasterMix 400 µL-20℃. Avoid freeze/thaw cycle.E665905BRNase-Free Water 2×1 mL-20℃. Avoid freeze/thaw cycle. Product features1. Convenience: The ready to use reverse transcription Mix only requires the addition of RNA templates and water to initiate the reaction.2. Fast: Complete cDNA first strand synthesis in 15 minutes.3. High reverse transcription efficiency: The reverse transcription efficiency is above 90%.4. High sensitivity: PG level templates can also obtain high-quality cDNA.5. Read through complex templates: templates with high GC content and complex secondary structures.Matters needing attention1. During the operation, RNase contamination should be avoided to prevent RNA degradation or cross contamination during experiments. It is recommended that operators wear masks and disposable gloves, frequently change gloves, and use specialized instruments and consumables.2. The reverse transcription system is prepared on ice for operation to prevent RNA degradation. The MasterMix of the reagent kit should be stored at -20 ℃ as soon as possible after use, and repeated freeze-thaw should be avoided as much as possible.3.10 µ The reaction system can be used up to 1 µ G Total RNA, if the amount of template RNA is greater than 1 µ g. Please expand the reaction system proportionally.4. For RNA templates with complex secondary structures, it is recommended to incubate the template RNA at 65 ℃ for 5 minutes on ice before proceeding with the next step, followed by brief centrifugation.Operation steps1. Thaw the template RNA on ice; After thawing the components of the reagent kit at room temperature, immediately place them on ice. Before use, vortex shake and mix each solution, and centrifuge briefly before use.2. Prepare the reaction system according to the following table (please prepare the reaction solution on ice), vortex shake and mix well, briefly centrifuge, and collect the solution on the tube wall to the bottom of the tube. Reagent 10 µl Reaction system Final concentration RNA Template X µl 1 pg～0.5 µg ¹⁾ 5×EasyQuick RT MasterMix ²⁾ 2 µl 1× RNase-Free Water up to 10 µl /Attention:1) If the total RNA content is greater than 1 µ g, please expand the reaction system proportionally.2) 5 x EasyQuick RT MasterMix contains Oligo (dT), Random Prime, RNase Inhibitor, dNTP Mixture, EQ-RT Buffer, etc. 3. Incubate at 37 ℃ for 15 minutes. 4. Incubate at 85 ℃ for 5 seconds to inactivate reverse transcriptase.5. After a brief centrifugation, place it on ice for subsequent experiments. If it needs to be stored for a long time, please place it at -20 ℃... Read More | ProductsThis product is a high purity genomic DNA extract from 293T cells, agarose gel (0.7%) electrophoresis showed that the size of the DNA extract is more than 15Kb, and basically no degradation, the product is ultimately preserved in TE Buffer, which can be widely used in molecular biology ProductsThis product is a high purity genomic DNA extract from 293T cells, agarose gel (0.7%) electrophoresis showed that the size of the DNA extract is more than 15Kb, and basically no degradation, the product is ultimately preserved in TE Buffer, which can be widely used in molecular biology experiments, such as PCR, enzyme digestion, hybridization, microarray analysis, and other molecular biology experiments.The product was quantified using NanoDrop One at a concentration of 200 ng/µL.Preparation and precautions before useLong-term storage at -20˚C is recommended. Before use, the bottle should be removed from the refrigerator and equilibrated to room temperature and centrifuged before opening the cap for use. Samples should be restored to the sealed state as soon as possible after opening.How to use (take qPCR experiment as an example)1. Amplification template preparationThe 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. The samples were placed on ice at 4°C and set aside.2. Standard dilution: according to the following table, firstly dilute Human DNA Standard 1 (100ng/uL) with TE to make 5 different concentrations of standards 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 not used temporarily after preparation, it should be stored at 4℃ or on ice.styleCorresponding concentration (ng/µL)Minimum dilution volume (in µL)Std.11010 [100 ng/µL DNA Standard 1] + 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 preparationThe cryopreserved reagents to be used were completely thawed and mixed by inversion several times before preparation, and then briefly centrifuged and prepared for use. 20 µL of the base reaction system was as follows.The base reaction system for 20 µL was as follows:reagents20µL reaction system2×qPCRMix10µLPrimerMixXµLProbeMixXµLTemplate4µLddH2OMake up to 20 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.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.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, so please refer to the manual of the instrument or the specific requirements for the use of each fluorescent probe for the adjustment of the concentration during actual use.Prepare a sufficient amount of reaction system mixture as required. After the reaction system has been prepared and mixed thoroughly, add 16 µL per well to the reaction wells. Then add the prepared standard and diluted sample into the corresponding reaction wells, the volume of addition is 4µL/well. TE was added to the blank control tube, and the same amount of TE 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 following is an example of our GoldStar Probe Mixture reaction conditions, which should be improved and optimized according to the PCR product template, primer structure and target fragment size.movetemptimingcirculatepremutability95°C10min1denaturation95°C10sec55Annealing/Extension60°C30sec5Data analysis1. 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 not be lower than 0.98, and the slope should be between -3.1 and -3.6 when the Ct value is the vertical coordinate. If the parameters of the standard curve are unreasonable, it is recommended to repeat the experiment... Read More | Format:1-ComponentEnzyme:Horseradish peroxidase |