| Description | Earthworm Fibrinolytic Enzyme (Lumbrokinase) is a type of enzyme extracted and purified from earthworms that can dissolve fibrin. It is present in the digestive tract of earthworms. Earthworm Fibrinolytic Enzyme has a dual function: in addition to directly hydrolyzing fibrin, it can also activate Earthworm Fibrinolytic Enzyme (Lumbrokinase) is a type of enzyme extracted and purified from earthworms that can dissolve fibrin. It is present in the digestive tract of earthworms. Earthworm Fibrinolytic Enzyme has a dual function: in addition to directly hydrolyzing fibrin, it can also activate plasminogen into plasmin, thereby exerting an indirect fibrin-hydrolyzing effect. After animals take Earthworm Fibrinolytic Enzyme, obvious antithrombotic and thrombolytic effects are produced. Lumbokinase attenuates myocardial ischemia-reperfusion (I-R) injury through the activation of Sirt1 signaling, and thus enhances autophagic flux and reduces I-R-induced oxidative damage, inflammation and apoptosis.CharacteristicsThis product is a pale yellow to yellowish-brown powder; it has a fishy odor and is hygroscopic.IdentificationTake an appropriate amount of this product, add water to prepare a solution containing 0.5mg per 1ml, and determine according to the spectrophotometry (Appendix IV A of the 2000 Edition of Pharmacopoeia of the People's Republic of China, Volume II); there shall be a maximum absorption at a wavelength of 278nm.Take an appropriate amount of this product, add 0.9% sodium chloride solution to prepare a solution containing 10mg per 1ml as the test solution; use a No. 6 needle to take 1 drop of animal blood and place it at the bottom of a test tube. After 30 minutes, add 1ml of the test solution to the test tube, shake gently, and the blood clot shall dissolve within 60 minutes. Use 1ml of 0.9% sodium chloride solution as the blank, and perform the same operation; the blood clot shall not dissolve.TestsAcidity and Alkalinity: Take this product, add water to prepare a solution containing 1mg per 1ml, and determine according to the method (Appendix VI H of the 2000 Edition of Pharmacopoeia of the People's Republic of China, Volume II); the pH value shall be 6.0 - 8.0.Clarity and Color of Solution: Take an appropriate amount of this product, add water to prepare a solution containing 1mg per 1ml; the solution shall be clear. If colored, it shall not be darker than the Yellow No. 2 Standard Color Solution (Method 1 of Appendix IX A of the 2000 Edition of Pharmacopoeia of the People's Republic of China, Volume II).Loss on Drying: Take an appropriate amount of this product, use phosphorus pentoxide as a desiccant, dry it under reduced pressure at 60℃ to constant weight; the weight loss shall not exceed 5.0% (Appendix VIII L of the 2000 Edition of Pharmacopoeia of the People's Republic of China, Volume II).Determination of Specific ActivityTiter DeterminationReagents0.01mol phosphate buffer (pH 7.8): Take 3.58g of disodium hydrogen phosphate, add water to dissolve and dilute to 1000ml as Solution A; take 0.78g of sodium dihydrogen phosphate (NaH₂PO₄·2H₂O), add water to dissolve and dilute to 500ml as Solution B; mix Solutions A and B until the pH value reaches 7.8.Working Solution: Mix 0.01mol/L phosphate buffer (pH 7.8) and 0.9% sodium chloride solution at a ratio of 1:17.1.5% Agarose Solution: Take 1.5g of agarose, add 100ml of the working solution, and heat to dissolve.Fibrinogen Solution: Take an appropriate amount of fibrinogen, add the working solution to prepare a coagulable protein solution containing 1.5mg per 1ml.Thrombin Solution: Take thrombin, add 0.9% sodium chloride solution to prepare a solution containing 1 BP unit per 1ml.Preparation of Standard Solution: Take the lumbrokinase standard, add 0.9% sodium chloride solution to prepare solutions with concentrations of 10,000, 8,000, 6,000, 4,000, and 2,000 lumbrokinase units per 1ml respectively.Preparation of Test Solution: Take an appropriate amount of this product, add 0.9% sodium chloride solution to dissolve and dilute to a concentration within the range of the standard curve.Determination Method: Take 39ml of the fibrinogen solution, place it in a beaker, add 39ml of the 55℃ agarose solution and 3.0ml of the thrombin solution while stirring, mix immediately, and quickly pour into a plastic petri dish with a diameter of 14cm. Place it horizontally at room temperature for 1 hour, then punch holes. Precisely measure 10µl each of the lumbrokinase standard solutions of different concentrations and the test solution, spot them on the same petri dish respectively, cover the dish, and place it in a 37℃ incubator for 18 hours of reaction. After taking it out, use a caliper to measure the vertical diameters of the lysis zones. Take the logarithm of the number of units of the kinase standard as the abscissa and the logarithm of the product of the vertical diameters as the ordinate, calculate the regression equation, and substitute the logarithm of the product of the vertical diameters of the test sample into the regression equation to calculate the titer units of the test sample. Both the standard and the test sample shall be tested in duplicate, and the average value shall be used for calculation.Protein ContentTake about 20mg of this product, weigh it accurately, determine according to the nitrogen determination method (Method 2 of Appendix VII D of the 2000 Edition of Pharmacopoeia of the People's Republic of China, Volume II), multiply the result by 6.25 to get the protein content in the test sample, and calculate the milligrams of protein per 1mg of the test sample.Specific ActivityCalculate the specific activity according to the following formula:... Read More | A general purpose purified albumin, suitable for Westerns, enzyme systems and as a protein supplement | Inquire | 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 | Reverse transcriptases are enzymes encoded in retroviruses viral genome. The enzyme is responsible for transcription of the viral RNA to produce a dsDNA that can be inserted into the host genome.Reverse transcriptases are multifunctional enzymes. These enzymes exhibit an RNA and DNA directed Reverse transcriptases are enzymes encoded in retroviruses viral genome. The enzyme is responsible for transcription of the viral RNA to produce a dsDNA that can be inserted into the host genome.Reverse transcriptases are multifunctional enzymes. These enzymes exhibit an RNA and DNA directed polymerase activity. In addition reverse transcriptases catalyze the degradation of RNA in an RNA-DNA hybrid. The exonucleolytic activity proceeds in a 5' ---> 3' direction. The RNA or DNA directed activity requires a template (RNA or DNA) and a primer. The following is a schematic illustration of the reaction:Unit definition: One unit incorporates 1 nanomole of tritiated dTMP into acid insoluble productsusing poly(A)•oligo(dT) 12-18 as the template-primer in 20 minutes at 37° C.ApplicationsHIV reverse transcriptase is used for research on the AIDS primer. However it can be substituted for AMV reverse transcriptase, which is mainly used to transcribe mRNA into double stranded cDNA, that can be inserted into prokaryotic vectors. The enzyme can also be used with either single stranded DNA or RNA templates to make probes for use in hybridization experiments. It can be used for labeling the termini of DNA fragments with protruding 5' termini. The enzyme can also be used to sequence DNAs by the dideoxy chain termination method of Sanger when the Klenow fragment of E. coli DNA polymerase I, or the T7 DNA polymerase yield unsatisfactory results.Reagents0.05 M Tris, pH 8.3, containing 0.008 M MgCl21 mg/ml polyadenylic acid in water (poly A)DNA primer:Oligo d(T)12-181 µ mole dTTP/mL stock solution[methyl-3H]-Thymidine 5'-triphosphate (3H-dTTP)dTTP-3H-dTTP working mix: Add 1-2 µL 3H-dTTP per mL of 100 nmol/mL dTTP in order to obtain 1 to 1.5 x 105 cpm/mL1% bovine serum albumin10% perchloric acid1% perchloric acidBuffer substrate reaction mixture: Prepare fresh, immediately before use:For each 1mL of reaction mixture required mix:0.7 mL Tris/HCl, pH 8.3, 0.008M MgCl20.3 mL 1 mg/mL poly(A) RNA template0.005 mL 0.02 mg/mL oligo d(T)12-18 DNA primer0.02mL 1% BSAEnzymedilute as needed wtih 0.05M Tris/HCl, pH 8.3, 0.008M MgCl2 containing 0.1 mg/mL (1%) BSAProcedurePipette into each tube as follows:Buffer substrate mix：0.1 mLdTTP-3H3-dTTP：0.1 mLEnzyme：5-10 µLIncubate 20 minutes at 37° C. Stop reaction by adding 1 ml 10% cold perchloric acid. Filter through 0.2µ manifold filters used with Millipore vacuum manifold. Wash four times using 2mL 1% cold perchloric acid/wash. Transfer filter to scintillation vials. Add 2mL Cellosolve (or 2-methoxyethanol) to dissolve filter. Filters become opaque upon addition of Cellosolve. Make sure filters are dissolved before proceeding. Add 10mL scintillation cocktail and count.Calculation... Read More |