39 results about "Phenylmethylsulfonyl Fluoride" patented technology

The invention discloses an evaluation method for the degree of thermal denaturation of macromolecular collagen by using a pepsin process. The method is characterized by comprising the following steps: (1) selection of a raw material: a step of selecting leftovers of aquatic product processing-fishskin as the raw material; (2) immersion and dissolving: a step of adding acetic acid with a concentration of 0.01 to 1.0 mol / L into the raw material to obtain a collagen solution; (3) heat pretreatment: a step of heating the collagen solution, shaking the solution every five minutes during heating and carrying out rapid cooling after heating is finished; (4) enzymatic hydrolysis: a step of adding pepsin into the collagen solution, carrying out a heat reaction and adding phenylmethylsulfonyl fluoride after the heat reaction is finished so as to terminate the reaction; and (5) preparation of an electrophoresis sample and electrophoresis. The invention has the following advantages: used equipment is simple; more comprehensive and visual reflection of the denaturation state of protein is realized.

The invention discloses an apple protein extraction method and a related protein extracting solution. The disclosed protein extraction method includes the steps that plant tissue and / or organs are smashed, the protein extracting solution is added and reacts for 3-7 minutes at the temperature of 0-6 DEG C, impurities are removed to obtain protein; solvent of the protein extracting solution is water, the protein extracting solution contains solutes of 100 mM of tri-hydroxymethyl aminomethane, 50 mM of ethylenediaminetetraacetic acid, 50 mM of potassium chloride, 25 mM of sodium tetraborate, 1 mM of dithiothreitol, 2 mM of phenylmethylsulfonyl fluoride, 1%(volume percentage concentration) polyethylene glycol octylphenol ether, 5% (volume percentage concentration) beta-mercaptoethanol and saccharose of 25 mg / mL, and the pH value is 7.5-8.0. By means of the protein extraction method, impurities such as polyphenol and pigment can be effectively removed, and when obtained protein is quantitatively analyzed through an iTRAQ technology, the number of the obtained protein can be larger than 2000.

The invention provides an extraction method of biological membrane proteins and a preparation method of an electrophoresis sample. The extraction method comprises the following steps: pretreating a biological membrane sample to obtain a precipitate; mixing the precipitate with a pyrolysis solution and a phenylmethylsulfonyl fluoride solution, and performing ultrasonic pyrolysis under an ice bath condition to obtain a sample pyrolysis solution; and shaking and centrifuging the sample pyrolysis solution, taking a supernatant, and treating the supernatant by adopting a TCA-acetone precipitation method, thereby obtaining a protein sample. The preparation method comprises the following steps: dissolving the protein sample by using a dissolving solution, and sequentially performing ultrasonic treatment, vortex oscillation and centrifugation, and taking a supernatant to obtain the electrophoresis sample. According to the methods provided by the invention, the pyrolysis solution containing various detergents is adopted and is combined with the ice bath ultrasound to break walls of microbial cells, and meanwhile, the TCA-acetone precipitation method is adopted to purify the protein, so that whole proteins in the biological membrane sample can be effectively extracted, and the masking action of humus to the proteins can be reduced to ensure that the analysis result of electrophoresis is clear.

The invention discloses a vaginal substrate material and a preparation method thereof, wherein a vaginal tissue mucous layer of a pig or a cow is taken as a carrier, and is subjected to the following treatment: a, carrying out shocking treatment by adopting deionized water solution containing phenylmethylsulfonyl fluoride or Tris solution; b, carrying out shocking treatment in a water solution containing lauryl sodium sulfate or Tris-HCl buffer solution; c, carrying out shocking treatment by adopting Tris-HCl buffer solution containing Triton X-100; d, carrying out shocking treatment in Tris-HCl buffer solution containing deoxyribonuclease and ribonuclease; and e, carrying out disinfection and freeze-drying. The vaginal substrate material provided by the invention has the biological activity and the biological mechanical property more suitable for pelvic floor repairing or vagina reconstruction.

The invention discloses a kit and a method for content determination of acetyl coenzyme A. The kit comprises a reagent I, a reagent II, a reagent III, a reagent IV and a reagent V, wherein the reagent I is prepared from Tris-HCl, polyvinylpyrrolidone, mercaptoethanol, phenylmethylsulfonyl fluoride and glycerinum; the reagent II is prepared from malic dehydrogenase; the reagent III is prepared from citrate synthase; the reagent IV is prepared from malic acid and an oxidation type coenzyme I; and the reagent V is prepared from Tris-HCl. The malic dehydrogenase can be used for catalyzing malic acid and the oxidation type coenzyme I to generate oxaloacetic acid and a reduced coenzyme I; the citrate synthase is used for catalyzing acetyl coenzyme A and oxaloacetic acid to generate citric acid and a coenzyme A; by virtue of a coupled reaction of the malic dehydrogenase and the citrate synthase, the content of the acetyl coenzyme A and the generation rate of the reduced coenzyme I are in a direct proportion; the climbing speed of the light absorption value at 340nm reflects the content of the acetyl coenzyme A. The kit disclosed by the invention is simple and convenient to operate, high in detection sensitivity and high in recovery rate, and the cost of the reagents is remarkably lowered. The detection steps are further simplified, so that the kit is simpler and more convenient and efficient to test.

The invention discloses a production method of polyvinylidene fluoride. The production method comprises the following steps of (1) adding 0.9 part by weight of hydrogen peroxide and 1.2 parts by weight of an inorganic alkali solution into a reaction kettle, uniformly mixing, then, adding 14.5 parts by weight of a trifluorotrichloroethane solvent and 1.8 parts by weight of a phenylmethylsulfonyl fluoride solution, and reacting at the temperature of 5 DEG C below zero for 25 minutes by stirring; (2) introducing 3.2 parts by weight of a 2-hexyl-1-decanol solution, 69 parts by weight of vinylidene fluoride monomers and 1.8 parts by weight of n-butyl cyclopentane, reacting at the temperature of 28 DEG C by stirring, and stopping reacting when the pressure is reduced to be smaller than 0.23MPa; and (3) filtering, washing and drying to obtain powdery polyvinylidene fluoride. Polyvinylidene fluoride prepared by using the production method is favorable in thermal stability and processibility and capable of meeting the requirements of production processes such as injection molding, extruding and film drawing.

The invention relates to a high-temperature Daqu leaching liquor macro-protein extraction and purification method. The method comprises the following steps: crushing high-temperature Daqu, spraying sterile water and evenly mixing, storing in a biological culture tank, performing activated culturing for later use by adopting a stage temperature rise method; adding an acetic acid-sodium acetate buffer solution and a phenylmethylsulfonyl fluoride solution, shaking and blending evenly, and then leaching overnight, filtering and centrifuging to obtain high-temperature Daqu leaching liquor; sequentially adding a TCA acetone solution, an ammonium acetate methanol solution, an acetone solution, a Tris satured phenol / lauryl sodium sulfate buffer solution, an ammonium acetate methanol solution, and a methanol and acetone solution to obtain sediment A, sediment B, phenol layer, sediment C, sediment D and high-temperature Daqu macro-protein samples; adding a sample lysate, performing ice bath ultrasonic for hydrotropy, and centrifuging the sediments to obtain a high-temperature Daqu macro-protein sample solution. The high-temperature Daqu macro-protein sample solution prepared by adopting the method can be applied to two-dimensional electrophoresis of the high-temperature Daqu macro-protein, to obtain high-resolution and high-repeatability two-dimensional electrophoretogram.

The invention discloses a preparation method of a polyvinylidene fluoride separation membrane. Polyvinylidene fluoride is taken as a membrane raw material; and the preparation method comprises following steps: 1) by weight, 1 to 2 parts of hexyldecanol, 0.8 part of hydrogen peroxide, and 1.1 parts of an inorganic base solution are delivered into a reaction vessel for uniform mixing, 1.6 parts of poly(ethylene glycol) monooleate, 14.5 parts of dichlorodifluoromethane solvent, and 1.8 parts of phenylmethylsulfonyl fluoride solution are subjected to 25min of reaction at -6 DEG C with stirring; 2) 1.3 parts of sodium oleoyl amino fatty acid, 2.2 parts of a hexyldecanol solution, 59 parts of vinylidene fluoride monomer, and 1.8 parts of methylcyclopentane are delivered into the reaction vessel, an obtained mixture is subjected to reaction at 18 DEG C with stirring, and reaction is stopped when pressure is reduced to be lower than 0.23MPa; and 3) an obtained product is filtered, washed, and dried so as to obtain polyvinylidene fluoride powder. The polyvinylidene fluoride separation membrane possesses corrosion resistance, heat resistance, cold resistance, oxidation resistance, and weather resistance.

The invention discloses an antibacterial aqueous paint used for wood furniture and a use method of same. The paint is composed of: modified alkyd resin, phenylated amino resin, 355-3 medium oil alkyd resin, ultrafine zirconium dioxide, calcium lignosulfonate, polyvinyl alcohol, polyamide wax slurry, phenylmethylsulfonyl fluoride, emulsified silicon oil, heavy aromatics, butadiene-vinyl pyridine latex and water. Compared with the prior art, a problem of poor water resistance of aqueous paints is solved by preparing the modified alkyd resin through a novel polymerization process to improve the physical and mechanical performance of the alkyd resin and compounding the alkyd resin with other raw materials. The paint is safe and environment-friendly, can form a stable film structure on the surface of a sheet material, and is significantly improved in the comprehensive physical and mechanical performance than the aqueous paints in the prior art. The paint has long-acting antibacterial effect and is suitable for being promoted.

The invention discloses a method for keeping phytoferritin stable, and belongs to the technical field of stabilizing functional food. Phenylmethylsulfonyl fluoride is added to a phytoferritin solution, so that the concentration of the solution is between 0.1 and 9 mmol / L to inhibit the degradation of the phytoferritin. The addition of the phenylmethylsulfonyl fluoride can keep good stability of the phytoferritin, so the function of iron supplementation activity is exerted effectively; and the method also increases the yield of protein, prolongs the storage time for the protein, and is favorable for wide application of the phytoferritin.

The invention provides a preparation method of a greater omentum acellular matrix and a construction method of cartilage tissue. The preparation method comprises the following steps: cleaning a greater omentum, performing freezing and thawing cycle treatment so as to obtain a first intermediate, adding the first intermediate into a solution which contains trypsin and phenylmethylsulfonyl fluorideand has a pH value of 7.8-8.2, maintaining the intermediate in the solution for a first preset period of time so as to obtain a second intermediate, performing degreasing treatment on the second intermediate, performing cleaning so as to obtain a third intermediate, adding the third intermediate into a solution which contains trypsin and phenylmethylsulfonyl fluoride and has a pH value of 7.8-8.2,maintaining the intermediate in the solution for a second preset period of time so as to obtain a fourth intermediate, performing tissue cell removal treatment on the fourth intermediate, performingcleaning so as to obtain a fifth intermediate, and performing drying and sterilization treatment on the fifth intermediate, so as to obtain a greater omentum acellular matrix material. The greater omentum acellular matrix prepared by using the preparation method provided by the invention is particularly applicable to regeneration culture of the cartilage tissue.

The invention discloses an extraction method of non-denatured protein and extraction liquid specially used by the method. The extraction method comprises a step a2): extracting the non-denatured protein in a sample by adopting the extraction liquid, wherein the extraction liquid contains sucrose, NaCl, L-sodium ascorbate, digitonin, dithiothreitol, phenylmethylsulfonyl fluoride and a protease inhibitor. The extraction method provided by the invention is easy and convenient to operate, a reagent is mild and economic, high-quality non-denatured protein can be efficiently extracted, the method lays a foundation and provides technical support for subsequently performed adverse plant proteomics research, and the method has a certain guiding significance for extracting recalcitrant tissue protein of plants living in an extreme environment. The method has a significant application value.

The invention relates to the technical field of biology and particularly relates to a rapid-detection kit for alpha-Gal antigens, an use method and application thereof. The invention provides a rapid-detection kit for the alpha-Gal antigens. The kit comprises a solid-phase alpha-Gal antigen coating porous plate, an alpha-Gal antigen standard substance, anti-rat-Gal IgM antibodies, goat-anti-rat IgM antibodies labeled with horseradish peroxidase, a hydrogen-peroxide developing solution, a TMB (Tetramethyl Benzidine) developing solution, a developing stopping solution, a tissue lysis solution, phenylmethylsulfonyl fluoride, concentrated washing liquor, a sample diluting solution, a Gal antigen positive biological material reference and a Gal antigen negative biological material reference. The rapid-detection kit provided by the invention has the beneficial effects that the detecting specificity to the alpha-Gal antigens reaches 100%, and 31.25ng / ml Gal antigen standard substance (the lowest detection limit) can be detected by sensitivity.

The invention discloses a porous hollow polyvinylidene fluoride fiber membrane. Polyvinylidene fluoride is taken as raw materials of the membrane and prepared through the following steps that 1, 1-2 parts by weight of carboxymethylcellulose, 2.8 parts by weight of hydrogen peroxide and 3.6 parts by weight of an inorganic alkali solution are put into a reaction kettle to be mixed evenly, 0.8 part by weight of lauryl sodium sulfate, 18.5 parts by weight of pentafluoroethane solvent and 0.8 part by weight of a phenylmethylsulfonyl fluoride solution are put into the reaction kettle, and reacting under stirring is performed for 45 minutes at minus 7 DEG C; 2, 3.4 parts by weight of glycerol monostearate, 6.2 parts by weight of a sodium tripolyphosphate solution, 69 parts by weight of vinylidene fluoride monomers and 1.9 parts by weight of 2-methyl-1-ethyl cyclopentane are put into the reaction kettle, reacting under stirring is performed at 23 DEG C, and reacting is stopped when the pressure is decreased to below 0.23 MPa; 3, filtering, washing and drying are performed, and then the powdery polyvinylidene fluoride is obtained. The porous hollow polyvinylidene fluoride fiber membrane has the corrosion resistance, heat resistance, cold resistance, oxidation resistance and weather resistance.

The invention provides a method for forecasting metastasis and recurrence of liver cancer after the operation of a primary liver cancer patient which comprises the followig concrete steps: protein extraction reagent is prepared by dissolving 3-[(3-cholanidopropyl)dimethylammonio]-1-propanesulfonate, dithiothreitol, ethylene diamine tetraacetic acid, phenylmethylsulfonyl fluoride, gastric inhibitory polypeptide and leupeptin in phosphate buffered solution with a pH value ranging from 7.0 to 7.5; a frozen liver cancer surrounding tissue is put in liquid nitrogen and smashed into powder, and a protein extraction reagent is added into the mixture which is then blended in a mixer; ultrasound treatment and centrifugation are carried out on the mixture; supernate is sucked and stored at the temperature ranging from 20 DEG C to 80 DEG C; and the concentrations of interleukin-2, interleukin-15 and interleukin-5 of the supernate are detected, for purpose of forecasting the metastasis and recurrence as well as survival or death after the operation of a liver cancer patient. The concentrations of the interleukin-2, the interleukin-15 and the interleukin-5 are obviously superior to the existing clinical indicator that is used for forecasting the recurrence and the survival of the liver cancer patient.

The invention provides a method for improving the expression quantity of a pyrethroid degeneration enzyme. The method comprises the following steps: preparing and producing strain escherichia coli (E.coli) BLR (DE3) / pET-28a-est825; culturing seeds; carrying out fermentation culture: cooling a mixed induction solution of lactose and isopropylthio-galactoside (IPTG) (at the molar concentration ratio of 7 to 3) to 30 DEG C to 35 DEG C and starting to induce, wherein a mixed material supplementing solution of glycerin and yeast powder is continually added in a flowing way in an induction process, the concentration of the glycerin in a fermentation solution is kept to be 1g / L to 2g / L, and induction time lasts for 6h to 7h; finally, preparing the strain into a fungal suspension solution with the mass concentration of 15 percent to 20 percent by utilizing a phosphate buffering solution containing 0.005mol / L of phenylmethylsulfonyl fluoride and having the pH (Potential of Hydrogen) of 6.5 to 7.0, and carrying out ultrasonic crushing, centrifuging and freeze-drying to obtain high-enzyme-activity degeneration enzyme powder.

The invention discloses an activity detection method for stem cell telomere repair active substances with good stability, which is characterized by comprising the following steps: S1, preparing a cell lysis solution, and performing lysis on mesenchymal stem cells to extract target protein; s2, preparing a reverse transcription reaction solution, and carrying out a reverse transcription reaction by adopting a PCR instrument; s3, preparing a QPCR reaction solution, and carrying out a fluorescent quantitative reaction by adopting a QPCR instrument; and S4, judging the relative activity of the telomere repairing active substance. The phenylmethylsulfonyl fluoride, the beta-mercaptoethanol and the dithiothreitol are compounded and added into the cell lysis solution, and the volume ratio of the phenylmethylsulfonyl fluoride, the beta-mercaptoethanol and the dithiothreitol in the cell lysis solution is limited, so that the cell lysis and reaction efficiency and quality are effectively improved; the time required for detecting the activity of the mesenchymal stem cell telomere repair active substance is fully shortened, the stability of the whole detection method is obviously improved, and the accuracy is higher.

The invention discloses a method for extracting vegetable tallow raft. The method comprises the following steps: (1) the plasma membrane is split to obtain lysis solution; (2) the density gradient centrifugation is carried out on the lysis solution in the step (1) to obtain the plant raft. The method uses dithiothreitol (DTT) to reduce the disulfide linkage, thus causing that the protein is fullydissolved. But phenylmethylsulfonyl fluoride (PMS) can effectively prevent the degradation of the protein in the extraction process. The method for extracting the plant raft has the advantages of being fast, simple and practicable and obtaining the tallow raft with higher quality and better repeatability. The method can play important role in the research of the vegetable tallow raft and has veryhigh practical using value.

The invention discloses a preparation method of recombinant grass carp interleukin-6 active protein. The method comprises the following steps of S1, culturing BL21(DE3) escherichia coli containing expression plasmid pET-30 / gcIL-6, and inducing protein expression for 15-25 hours; S2, collecting fermentation liquor, performing centrifugation, discarding supernate, resuspending thalli, and performingfreezing and thawing for 10-20 hours; S3, adding a phenylmethylsulfonyl fluoride solution into bacterial liquid, carrying out ultrasonic treatment, then performing centrifugation, and collecting precipitate, namely an inclusion body; S4, carrying out oscillation washing on the inclusion body by using a washing buffer solution, and then performing centrifugation; S5, carrying out oscillation washing on the inclusion body by using 1% Triton X-114, and performing centrifugation; S6, carrying out oscillation washing on the inclusion body by using 3M urea, and performing centrifugation; S7, dissolving the inclusion body by using a binding buffer solution containing 6M guanidine hydrochloride, performing centrifugal dissolving for 5-15 minutes, and reserving supernate; S8, purifying protein; S9, dropwise adding the purified protein into a folding buffer solution, then performing stirring for 10-20 hours, and then performing filtration and centrifugation to obtain a protein solution; and S10, putting the protein solution into a phosphate buffer solution, and performing centrifugation to obtain the recombinant grass carp interleukin-6 active protein. Through the method, a large amount ofthe recombinant grass carp IL-6 protein with high biological activity can be obtained.

The invention discloses a protein stabilizer and a preparation method thereof. The protein stabilizer comprises the following raw materials: high molecular weight hyaluronic acid, medium molecular weight hyaluronic acid, low molecular weight hyaluronic acid, oligomeric hyaluronic acid, acetylated hyaluronic acid, sodium polyacrylate, disodium ethylene diamine tetraacetate dihydrate, phenylmethylsulfonyl fluoride, magnesium chloride, polyethylene glycol monomethyl ether, sodium chloride or potassium chloride, ProCin 950, ProCin 300, bovine serum albumin, collagen, dipropylene glycol or glycerol, and a phosphate or Tris-HCl buffer solution. The quality guarantee period and stability of various proteins can be greatly prolonged by using a compounding technology of large and small molecular hyaluronic acid, gelatin and the like and a synergistic effect of macromolecules, preservatives and the like, the proteins, such as polystyrene boards, latex beads, magnetic particles, nitrocellulose membranes and the like, on various surfaces or in solutions can be greatly prolonged, and the stabilizers also reduce the background by eliminating non-specific binding of sample matrices and other assay ingredients.

The invention discloses a kit and a method for content determination of acetyl coenzyme A. The kit comprises a reagent I, a reagent II, a reagent III, a reagent IV and a reagent V, wherein the reagent I is prepared from Tris-HCl, polyvinylpyrrolidone, mercaptoethanol, phenylmethylsulfonyl fluoride and glycerinum; the reagent II is prepared from malic dehydrogenase; the reagent III is prepared from citrate synthase; the reagent IV is prepared from malic acid and an oxidation type coenzyme I; and the reagent V is prepared from Tris-HCl. The malic dehydrogenase can be used for catalyzing malic acid and the oxidation type coenzyme I to generate oxaloacetic acid and a reduced coenzyme I; the citrate synthase is used for catalyzing acetyl coenzyme A and oxaloacetic acid to generate citric acid and a coenzyme A; by virtue of a coupled reaction of the malic dehydrogenase and the citrate synthase, the content of the acetyl coenzyme A and the generation rate of the reduced coenzyme I are in a direct proportion; the climbing speed of the light absorption value at 340nm reflects the content of the acetyl coenzyme A. The kit disclosed by the invention is simple and convenient to operate, high in detection sensitivity and high in recovery rate, and the cost of the reagents is remarkably lowered. The detection steps are further simplified, so that the kit is simpler and more convenient and efficient to test.

The invention relates to a high-temperature Daqu leaching liquor macro-protein extraction and purification method. The method comprises the following steps: crushing high-temperature Daqu, spraying sterile water and evenly mixing, storing in a biological culture tank, performing activated culturing for later use by adopting a stage temperature rise method; adding an acetic acid-sodium acetate buffer solution and a phenylmethylsulfonyl fluoride solution, shaking and blending evenly, and then leaching overnight, filtering and centrifuging to obtain high-temperature Daqu leaching liquor; sequentially adding a TCA acetone solution, an ammonium acetate methanol solution, an acetone solution, a Tris satured phenol / lauryl sodium sulfate buffer solution, an ammonium acetate methanol solution, and a methanol and acetone solution to obtain sediment A, sediment B, phenol layer, sediment C, sediment D and high-temperature Daqu macro-protein samples; adding a sample lysate, performing ice bath ultrasonic for hydrotropy, and centrifuging the sediments to obtain a high-temperature Daqu macro-protein sample solution. The high-temperature Daqu macro-protein sample solution prepared by adopting the method can be applied to two-dimensional electrophoresis of the high-temperature Daqu macro-protein, to obtain high-resolution and high-repeatability two-dimensional electrophoretogram.

The invention discloses a cell lysis method for activity detection of stem cell telomere prolongation protein, which comprises the following steps: (1) collecting to-be-detected cells resuspended by sodium chloride injection, centrifuging to remove supernatant, and leaving cell precipitate; (2) diluting a phenylmethylsulfonyl fluoride solution, beta-mercaptoethanol and a lysis aid by using a cell lysis buffer solution, adding the three diluted solutions into the cell lysis buffer solution, and uniformly mixing in a vortex manner to obtain a cell lysis solution; (3) adding a cell lysis solution into the cell precipitate, then carefully blowing and beating the cells up and down by using a pipette until the cells are uniformly dispersed, and splitting the cells on ice; and (4) centrifuging the lysed cells at low temperature, sucking supernate, and transferring the supernate into a new centrifuge tube to obtain a cell lysis sample. The cell lysis method provided by the invention has good cell lysis effect and telomere extension protein activity detection accuracy, and effectively reduces the telomere extension protein activity detection time.

The invention discloses a magnetic bead method nucleic acid extraction reagent formula capable of effectively improving virus nucleic acid recovery efficiency. Comprising urea, thiourea, thiothreitol, ethylenediamine tetraacetic acid, cholamidopropyl, tromethamine, ethyl phenyl polyethylene glycol, polyethylene glycol octyl phenyl ether, phenylmethylsulfonyl fluoride, sodium chloride, sodium fluoride, guanidine hydrochloride, guanidine isothiocyanate, protease K, sodium deoxycholate, distilled water, a protease inhibitor, a phosphatase inhibitor and sodium orthovanadate. The preparation method mainly comprises the following steps: adding guanidine hydrochloride into urea to achieve a solubilizing effect on amino acid, adding protease K to cut carboxyl-terminal peptide bonds of aliphatic amino acid and aromatic amino acid, adding a protease inhibitor to effectively inhibit various asparaginic acid proteases, and adding a phosphatase inhibitor to inhibit various asparaginic acid proteases. Alkaline phosphatase and tyrosine phosphatase can be inhibited, the recovery efficiency of viral nucleic acid can be effectively improved, the working efficiency is improved, and the method is convenient and practical.

The invention provides a method for improving the expression quantity of a pyrethroid degeneration enzyme. The method comprises the following steps: preparing and producing strain escherichia coli (E.coli) BLR (DE3) / pET-28a-est825; culturing seeds; carrying out fermentation culture: cooling a mixed induction solution of lactose and isopropylthio-galactoside (IPTG) (at the molar concentration ratio of 7 to 3) to 30 DEG C to 35 DEG C and starting to induce, wherein a mixed material supplementing solution of glycerin and yeast powder is continually added in a flowing way in an induction process, the concentration of the glycerin in a fermentation solution is kept to be 1g / L to 2g / L, and induction time lasts for 6h to 7h; finally, preparing the strain into a fungal suspension solution with the mass concentration of 15 percent to 20 percent by utilizing a phosphate buffering solution containing 0.005mol / L of phenylmethylsulfonyl fluoride and having the pH (Potential of Hydrogen) of 6.5 to 7.0, and carrying out ultrasonic crushing, centrifuging and freeze-drying to obtain high-enzyme-activity degeneration enzyme powder.

The invention discloses a method for keeping phytoferritin stable, and belongs to the technical field of stabilizing functional food. Phenylmethylsulfonyl fluoride is added to a phytoferritin solution, so that the concentration of the solution is between 0.1 and 9 mmol / L to inhibit the degradation of the phytoferritin. The addition of the phenylmethylsulfonyl fluoride can keep good stability of the phytoferritin, so the function of iron supplementation activity is exerted effectively; and the method also increases the yield of protein, prolongs the storage time for the protein, and is favorable for wide application of the phytoferritin.

The invention discloses a liquid chromatography-mass spectrometry measuring method of an acetyl coenzyme A in animal livers. The method specifically comprises the steps that firstly, the fresh animallivers are taken, and blood in the animal livers is washed off; then the liver sample is mixed with pretreatment liquid, homogenizing is carried out, and thus tissue homogenate is prepared, wherein the pretreatment liquid is a perchloric acid aqueous solution containing dithiothreitol and phenylmethylsulfonyl fluoride; an internal standard substance is added into the homogenate and mixed evenly, then centrifugation is carried out, and supernatant is taken as a sample to be detected; and finally, the sample is sent to a liquid chromatography-mass spectrometry detector to be detected, data are collected, and after standard curve regression calculation, the concentration of the acetyl coenzyme A in the animal livers is obtained. Compared with an existing enzyme-linked immunoassay labeling method, the liquid chromatography-mass spectrometry method adopted by the invention is rapid, reliable and simple in operation step and suitable for measuring the content of the acetyl coenzyme A in thelivers in batches.

The invention discloses an extraction method of non-denatured protein and extraction liquid specially used by the method. The extraction method comprises a step a2): extracting the non-denatured protein in a sample by adopting the extraction liquid, wherein the extraction liquid contains sucrose, NaCl, L-sodium ascorbate, digitonin, dithiothreitol, phenylmethylsulfonyl fluoride and a protease inhibitor. The extraction method provided by the invention is easy and convenient to operate, a reagent is mild and economic, high-quality non-denatured protein can be efficiently extracted, the method lays a foundation and provides technical support for subsequently performed adverse plant proteomics research, and the method has a certain guiding significance for extracting recalcitrant tissue protein of plants living in an extreme environment. The method has a significant application value.

The application provides a carboxylesterase inhibitor preparation for drug metabolism detection, the carboxylesterase inhibitor preparation is composed of phenylmethylsulfonyl fluoride and calcium chloride, the carboxylesterase inhibitor preparation It has a high inhibition rate and is not affected by the anticoagulant heparin, and can be combined with heparin to make a blood preservation agent.