| 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 | Product contentF665774Component5 mLStorageF665774A2×Fast Probe Mixture5×1 mL-20℃. Avoid freeze/thaw cycle.F665774B50×High ROX200 µL-20℃. Avoid freeze/thaw cycle.F665774CddH2O5×1 mL-20℃. Avoid freeze/thaw cycle.Product IntroductionFast Probe Mixture is a preProduct contentF665774Component5 mLStorageF665774A2×Fast Probe Mixture5×1 mL-20℃. Avoid freeze/thaw cycle.F665774B50×High ROX200 µL-20℃. Avoid freeze/thaw cycle.F665774CddH2O5×1 mL-20℃. Avoid freeze/thaw cycle.Product IntroductionFast Probe Mixture is a pre-mixed system for real-time fluorescence PCR by probe method (TaqMan, Molecular Beacon, etc.), with a concentration of 2×, including Fast Taq DNA Polymerase, PCR Buffer, dNTPs, Mg2+ and so on, which is easy and convenient to operate. It is mainly used for the detection of genomic DNA target sequence and cDNA target sequence after RNA reverse transcription. The Fast Taq DNA Polymerase contained in this product can effectively reduce the non-specific amplification generated by the non-specific binding of primers and templates or primer dimerization at room temperature, and the activation of the enzyme only needs to be incubated at 95 ℃ for 30 s. The whole PCR reaction process can save about 40 minutes compared with the ordinary reaction, which greatly shortens the reaction time of PCR. The combination of unique PCR buffer system and fast hot start enzyme effectively inhibits the generation of non-specific products and significantly improves the PCR amplification efficiency with stronger fluorescence signal, higher sensitivity and wider linear range. The product has a wide range of applications and can be used for both normal and rapid quantitative PCR programs.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 (F665766):Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96 and others.Instruments that require Low ROX calibration (F665768):ABI Prism7500/7500 Fast, QuantStudio®3 System, QuantStudio®5 System, QuantStudio®6 Flex System, QuantStudio®7 Flex System, ViiA 7 system. Stratagene Mx3000/Mx3005P, Corbett Rotor Gene 3000, and more.Instruments that require High ROX calibration (F665774):ABI Prism 7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, and others.matters needing attention1. Before use, please mix gently by turning up and down, avoid foaming as much as possible, and use after brief centrifugation.2. Avoid repeated freezing and thawing of this product, repeated freezing and thawing may degrade the product performance. This product can be stored for long term at -20℃, protected from light. If frequent use is required within a short period of time, it can be stored at 2-8℃.UsageThe following examples are conventional PCR reaction systems and reaction conditions, which should be improved and optimized according to the template, primer structure and target fragment size in actual operation.1.PCR reaction systemreagents50µl reaction systemfinal concentration2×Fast Probe Mixture25 µl1×Forward Primer, 10µM1µl0.2µM¹⁾Reverse Primer, 10µM1µl0.2µM¹⁾Probe, 10 µM1µl0.2µM²⁾Template DNA2µl³⁾ 50x Low ROX or High ROX(optional)⁴⁾1µl1×ddH₂Oup to 50µlNote: 1) Usually the primer concentration of 0.2µM can get better results, and 0.1-1.0µM can be used as a reference for setting the range. 2) The final 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 instruction manual of the instrument or the specific requirements of the use of each fluorescent probe for the adjustment of the concentration in actual use.(3) Usually the amount of DNA template is 10-100ng genomic DNA or 1-10ng cDNA as a reference. Since the templates of different species contain different copy numbers of target genes, the templates can be subjected to gradient dilution to determine the optimal amount of template to be used.(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.2. PCR reaction program:A two-step PCR reaction program is recommended, and this program is set up using the ABI 7500 Fluorescent Quantitative PCR Instrument as a reference.Note: 1) The enzyme used in this product must be pre-denatured at 95°C for 30s to achieve enzyme activation. Under this condition, most of the templates can be well unchained. For templates with high GC content and complex secondary structure, the pre-denaturation time can be extended to 1-4 minutes to allow the starting template to fully unchain.(2) It is recommended to use two-step PCR reaction program, if you do not get good experimental results due to the use of primers with lower Tm values, etc., you can try to carry out three-step PCR amplification, and the annealing temperature, please use the range of 56 ℃ - 64 ℃ as a setting reference... Read More | Inquire | Inquire | Inquire |