52 results about "Enzymatic digestion" patented technology

The invention belongs to the cross technical field of veterinary medicine and biomedicine, and relates to a construction and evaluation method of a dog spontaneous breast cancer organoid model, which specifically comprises the following steps: (1) obtaining dog spontaneous breast cancer tissues through operation or biopsy, separating tumor tissues under sterile conditions, and carrying out fine enzymolysis digestion treatment to obtain dog spontaneous breast cancer tissues; obtaining a single-cell suspension; and (2) inoculating the single-cell suspension into a three-dimensional culture system, carrying out three-dimensional culture by adopting a culture medium containing EGF, FGF and B27 in combination with a growth factor, promoting the growth and amplification of the breast cancer organs, and carrying out timely passage according to the growth condition so as to maintain the activity of the model. The model constructed by the invention completely retains genetic heterogeneity and pathological microenvironment of primary tumors, does not need gene editing, is simple and convenient to operate and low in cost, is particularly suitable for individual drug sensitivity detection of pet dogs, and provides an efficient and high-simulation experimental platform for dog breast cancer mechanism research and clinical precise medication.

This invention discloses a method for extracting high-purity PDRN from salmon testes and its application in improving skin condition, belonging to the field of bioactive ingredient extraction technology. Frozen salmon testis tissue is cleaned of blood vessels and fatty impurities, then cryogenically pulverized at -20~-25℃. The pulverized tissue is added to a pre-treatment dispersion buffer and homogenized under high pressure to obtain a DNA fragmentation solution. The precipitate is then separated by centrifugation. The precipitate is dispersed in a dedicated enzyme digestion buffer, and enzymatic digestion with protease and RNase A is performed to inactivate the enzyme. DNase I is added to the digestion solution for enzymatic cleavage. After the reaction, a stop solution is added to terminate the reaction, and the supernatant is separated by centrifugation. Ethanol is added to the supernatant for precipitation, and centrifugation yields PDRN from salmon testes. This invention, through specific buffer systems and enzymatic digestion conditions, increases the PDRN content by 40~60 kDa, allowing for better absorption and utilization by the skin, improving facial hydration and elasticity, effectively reducing facial wrinkles, and improving facial skin condition. It can be used to prepare functional cosmetics.

This invention discloses a method for inducing and culturing burdock stem cells and stem cells, belonging to the field of plant biotechnology. Using burdock roots as explants, the cambium is determined by staining, and the cambium is obtained using enzymatic digestion. The cambium is then inoculated into a specific ratio of induction solid medium and cultured in the dark to efficiently induce primary stem cells. These primary stem cells are then transferred to a proliferation solid medium for subculture and selection to obtain and maintain a vigorous, soft stem cell line. The solid stem cell line is further transferred to a liquid medium for shaking culture to establish a suspension stem cell line. This invention provides high induction efficiency, stable stem cell characteristics, and good reproducibility, offering a high-quality starting material and technical platform for in vitro preservation of burdock germplasm, large-scale production of secondary metabolites, and genetic transformation.

The invention belongs to a proteomics analysis technology in the technical field of biology, and particularly relates to a single-cell-level proteomics analysis system and analysis method for a mass spectrum system. The analysis system comprises a sample pretreatment optimization subsystem and a chromatography-mass spectrometry acquisition optimization subsystem, the purpose of the sample pretreatment optimization subsystem is to efficiently and stably prepare a peptide fragment mixture capable of being used on a mass spectrum machine from a trace cell sample. The sample loss is reduced by improving the protein extraction and enzymolysis efficiency of trace (nanogram-level) and single-cell-level protein samples. By optimizing mass spectrum data acquisition parameters, the identification depth and quantity of proteins are improved while the analysis time is shortened (the flux is improved). By adopting the analysis system disclosed by the invention, the accuracy and reproducibility of quantitative analysis of the micro sample proteome can be remarkably improved.

This invention discloses a PEG-mediated genetic transformation method for *A. anthracnose* protoplasts. Through the preparation, transformation, and transformation of *A. anthracnose* protoplasts, and the screening of transformants, the hygromycin B resistance gene fragment was successfully transferred into the *A. anthracnose* genome, achieving successful genetic transformation of *A. anthracnose* protoplasts. More importantly, this invention establishes a new enzymatic digestion system and improves the culture mechanism in protoplast preparation. During the culture and screening of transformants, a more suitable resistance concentration is used, balancing the stringency of screening with the survival of transformants. This improves transformation efficiency, shortens the overall transformation process, and establishes a more efficient and convenient genetic transformation system for *A. anthracnose* protoplasts.

The invention discloses a nucleotide sequence of PDRN / PN and a preparation method of PDRN / PN based on genetic engineering and microbial fermentation, and belongs to the technical field of biology. According to the method, the vibrio is adopted as a production chassis strain, and methods of pyrolysis crude extraction, enzyme digestion and magnetic bead selective separation are combined, so that the preparation of the multi-source PDRN / PN is realized, and the application range of the product is expanded. Compared with an escherichia coli system, the method has the advantages that the endotoxin pollution risk can be remarkably reduced, and the product safety is improved; compared with a yeast system, the production efficiency of PDRN / PN can be improved. Meanwhile, the method is simple and convenient to operate, is suitable for laboratory and pilot scale amplification, and has obvious industrial application potential.

This invention discloses a method for protein extraction and mass spectrometry pretreatment from methanogenic bacteria, belonging to the field of protein extraction technology. The method involves pretreating a methanogenic bacteria enrichment culture to remove inorganic salt ions and other impurities, resulting in a pretreated culture. Then, a lysis buffer composed of sodium dodecyl sulfate, Tris-HCl buffer, and a protease inhibitor is added to the pretreated culture, followed by ultrasonic disruption to obtain a cell lysate, from which a crude protein extract solution is obtained. Finally, the protein extract is extracted from the crude protein extract solution. This invention significantly improves protein extraction efficiency and peptide purity by optimizing the extraction process and subsequent enzymatic digestion, while preserving the methanogenic activity of the extracted protein as much as possible, providing more accurate and reliable sample support for downstream protein mass spectrometry analysis.

This invention belongs to the field of renal pathology testing technology and discloses a method for detecting type IV collagen expression in renal biopsy tissue, comprising the following steps: dewaxing and hydrating paraffin sections of renal biopsy tissue to obtain processed tissue sections; adding the processed tissue sections to a repair solution, heating to boiling, and then cooling to room temperature to obtain repaired tissue sections; adding protease to the repaired tissue sections for enzymatic digestion to obtain enzymatically digested tissue sections; adding a primary antibody to the enzymatically digested tissue sections for preliminary incubation, followed by adding a fluorescein-labeled secondary antibody against the primary antibody for further incubation. This method for detecting type IV collagen expression in renal biopsy tissue is applicable to cases where renal biopsy tissue lacks glomeruli or has too few glomeruli to detect type IV collagen expression. It has high specificity and can provide a new detection method for Alport syndrome in cases where renal biopsy tissue lacks glomeruli.

The application belongs to the technical field of genetic engineering. The application provides application of a thiol-oxidoreductase in promoting keratin enzymolysis and a method for promoting keratin enzymolysis. The thiol-oxidoreductase is applied to a keratin enzymolysis system, and the enzymolysis effect is obviously improved. Through testing, the enzymolysis effect of the thiol-oxidoreductase combined with a protease on keratin is not only obviously higher than the enzymolysis effect of a single protease, but also is better than the hydrolysis of keratin by a chemical reducing agent with the same concentration. The application retains the advantages of enzyme method "green chemistry" and is friendly to the environment.

The invention discloses a liquid chromatography-tandem mass spectrometry determination method for altrenogest in pig tissue, which comprises the following steps: sample pretreatment: enzymolysis treatment: weighing 1.0 g of pig tissue sample which is pig liver, pig muscle or pig skin adipose tissue; a protease K solution is added into the pig tissue sample, pig liver or pig muscle tissue is subjected to enzymolysis for 1 h in a dark water bath at the temperature of 37 DEG C, and pig skin adipose tissue is subjected to enzymolysis for 30 min in a dark water bath at the temperature of 50 DEG C; performing ultrasonic treatment for 2 minutes after enzymolysis, and cooling to room temperature; according to the method, the purification efficiency and the specificity are greatly improved, and the problem of matrix interference is effectively solved. The MIP-MNPs prepared by taking altrenogest as a template molecule can specifically adsorb altrenogest and metabolites thereof through spatial structure matching and functional group action, so that non-specific interference of protein, fat and steroid impurities in pig tissues is eliminated, and the ionization inhibition effect of a matrix on mass spectrometry detection is remarkably reduced.

The application discloses a DNA / RNA reagent kit based on a magnetic bead method and free of enzyme digestion, and belongs to the technical field of nucleic acid extraction and purification. The reagent kit comprises a lysis solution AL, protease K, a specific binding buffer CLB, amino magnetic bead suspension, silicon hydroxyl magnetic bead suspension and other components. The application solves the problems of nucleic acid degradation, incomplete removal of cross contamination and complicated process caused by enzyme digestion in the prior art, is suitable for automatic high-throughput processing, and has a wide application prospect in scientific research and clinical application.

This invention relates to the field of biodetection technology, specifically to a non-isolated digital multiplex detection method based on a DNA walker and TdT enzyme amplification. The method first utilizes coding microspheres with capture elements coupled to their surface to specifically bind to target miRNAs or proteins in the sample; then, specific guide DNA (gDNA) corresponding to the target molecule is released through enzymatic digestion or thermal denaturation; the gDNA then... Pf Under the action of Ago endonuclease, the DNA walker on the surface of the encoded microspheres is activated, cyclically cutting the orbital strand to generate multiple DNA fragments with 3'-hydroxyl ends. Finally, TdT enzyme is used to extend fluorescently labeled nucleotides at the hydroxyl ends, achieving in-situ signal amplification, and digital quantitative analysis is performed on the fluorescence signal of the encoded microspheres. This invention achieves high-sensitivity detection of multiple targets in a single reaction without chamber isolation, with detection limits for each target molecule reaching the fM or fg / mL level, providing an effective technical means for the early screening of major diseases such as Alzheimer's.

The invention discloses a construction and evaluation method of an Rb1 and Trp53 (p53) double-gene mutation mouse spontaneous lung cancer organoid model, and the construction method comprises the following steps: separating lung cancer tissues under a sterile condition, and carrying out fine enzymolysis digestion treatment to obtain a single-cell suspension; inoculating the single-cell suspension into a three-dimensional culture system, carrying out three-dimensional culture by adopting a culture medium containing EGF, FGF and B27 in combination with growth factors, promoting the growth and amplification of lung cancer organs, and carrying out timely passage according to the growth condition to maintain the activity of the model; the model constructed by the invention completely retains genetic heterogeneity and pathological microenvironment of the primary tumor, in-vitro gene editing is not needed, the operation is simple and convenient, the cost is low, and an efficient and high-simulation experimental platform is provided for lung cancer mechanism research and individualized drug screening.

The present disclosure relates to a method of digesting a sample comprising a protein, the method comprising: adding the sample to a device, the device containing a buffer and a solid support surface comprising a surface coating, wherein the surface coating immobilizes an enzyme while reducing undesired interactions between the sample and the solid support surface; immobilizing the enzyme on the surface coating for digesting a protein of the sample; and heating the sample to complete a heat-assisted digestion of the protein.

This application belongs to the field of genetic engineering technology and discloses human enterokinase light chain mutants and their applications. This application involves single-point mutations or multiple rounds of combined mutations based on the wild-type human enterokinase light chain -C112S mutant sequence. The obtained variants have higher specific activity compared to the parent, which is expected to increase unit yield, reduce the residual amount of protease in the final product, and lower product costs. Furthermore, these mutants exhibit high stability in the enzymatic digestion of peptides / proteases, such as liraglutide and smegglutide, GLP-1 analogs, reducing enzyme usage, increasing digestion efficiency, and decreasing the proportion of non-specific products, thereby lowering the production cost of peptide drugs and promoting the rapid development of related industries.

The application relates to the technical field of biological analysis, in particular to a cell surface N-sugar detection method based on enzymolysis and fluorescent labeling. The specific technical scheme comprises the following steps: using trypsin to perform enzymolysis treatment on cells to obtain glycoprotein; performing denaturation treatment on the glycoprotein, and then performing PNGase F enzyme cutting to obtain N-sugar chains; performing purification treatment on the N-sugar chains to obtain purified N-sugar chains; using 8-aminopyrene-1,3,6-trisulfonic acid to perform fluorescent labeling on the purified N-sugar chains to obtain fluorescently labeled N-sugar chains; and performing fluorescent detection on the fluorescently labeled N-sugar chains after purification treatment. The detection method greatly improves the extraction efficiency and detection sensitivity, and is low in cost and simple in steps.

The application belongs to the technical field of food detection, and provides a detection method of soluble dietary fiber in resistant dextrin. The method adopts amylodextrinase to perform enzymatic digestion on the resistant dextrin, adds water to prepare a test solution; glucose is used as a standard, high performance liquid chromatography is used for detection, and the content of the soluble dietary fiber in the resistant dextrin is obtained by an external standard method. The detection method is simpler than the existing standard method, greatly reduces the error sources, can guarantee the accuracy and precision of the detection results, shortens the detection time, saves manpower and economic cost, and can meet the detection needs of inspection supervision departments and enterprises.

The application belongs to the technical field of cosmetics, and particularly relates to a preparation method of a small molecule polypeptide of a leaf-eating plant and an amino acid solution and application thereof. The method obtains specific peptide segments by gradually enzymatically digesting proteins contained in the leaf-eating plant and by controlling pH values and enzyme types, so as to realize efficient enzymatic digestion and improve the purity of products, thereby laying a foundation for the application of the products in the field of cosmetics. The method of the application not only has safe and reliable production principles, but also improves the yield of the small molecule polypeptide of the leaf-eating plant, and solves the technical problem that cosmetic protein raw materials are difficult to penetrate the skin.

This invention discloses a uniform-size silk fibroin microsphere, its preparation method, and its application, comprising the following steps: taking a regenerated silk fibroin solution, digesting it with trypsin to obtain a modified silk fibroin precipitate, dissolving it in an aqueous solution of a β-sheet inhibitor to obtain a silk fibroin solution as the dispersed phase; emulsifying it using an external pressure membrane emulsifier to obtain an emulsion containing silk fibroin droplets; adding a cross-linking agent to the emulsion to carry out a cross-linking reaction, and then adding a crystallization inducing agent to induce crystallization to obtain primary silk fibroin microspheres; and freeze-drying under vacuum to obtain silk fibroin microspheres. This invention modifies silk fibroin by enzymatic digestion to retain silk fibroin fragments with high β-sheet content, exhibiting high structural stability; and uses membrane emulsification combined with a multiple crystallization induction method to improve the crystallinity of the microspheres, obtaining silk fibroin microspheres with uniform particle size and smooth surface.

The present disclosure provides methods for enriching, identifying and / or characterizing at least one host cell protein (HCP) impurity in a sample containing a viral vector. HCP impurities can be enriched by utilizing a bead-based peptide ligand library. The enriched HCP may then be subjected to enzymatic digestion, producing a peptide that can be identified by liquid chromatography-mass spectrometry (LC-MS) analysis to specifically identify and / or characterize the at least one HCP impurity.

Examination of a test sample to determine the succination level of test sample is described. Examination can be via protein hydrolysis in total succination determination or via enzymatic digestion of isolated proteins and determination of the presence or quantity of modified peptides. The methods can be utilized for determination of excessive succination of lymph system proteins, which can be utilized in prevention or early detection of autoimmune disorders. Methods can be utilized for test samples of subjects under treatment with dimethyl fumarate suffering from an autoimmune disorder. Methods can be utilized as a determination that treatment of the subject with DMF should be slowed or stopped.

The invention discloses an intestinal targeting probiotic emulsion gel capsule and a preparation method thereof. The preparation method comprises the following steps: firstly, preparing an acidic water phase containing probiotics and cationic polysaccharide, and preparing an oil phase containing a hydrophobic solid particle emulsifier and edible oil; the oil phase and the water phase are emulsified at a high speed, and stable emulsion gel is formed on an oil-water interface by utilizing an electrostatic crosslinking effect between the cationic polysaccharide and solid particles; further, edible colloid is used as a shell, and the emulsion gel inner core is packaged through a micro-fluidic technology to prepare the emulsion gel capsule. The emulsion gel disclosed by the invention can effectively resist a gastric acid environment, protect the activity of probiotics and realize targeted release in intestinal tracts by digesting an oil phase through lipase. The capsule is completely made of safe materials approved by FDA, has good stability, machinability and biocompatibility, remarkably improves the oral delivery efficiency of probiotics, and is suitable for the field of functional food and pharmaceutical preparations.

The invention discloses pure, soluble, and functional exoglycosidase enzymes that can be produced in high yield and cost-effective way using bacterial expression systems. Peptide tag, preferably HIS-tag is used for potential immobilization to ensure the best accessibility to the active sites of the enzymes targeting automation purposes and special workflows. The invention provides rapid enzymatic digestion performance both in aqueous phase and in immobilized form. Immobilized enzymes allow long term storage and ready to use pre-mixing. The inventive immobilization approach opens up the possibility for automation and for meeting special experimental needs where the immobilization of the enzymes is key.

The application belongs to the technical field of mass spectrometry, and particularly relates to a mass spectrometry detection method and application of intracellular blood coagulation factor X, which comprises the following steps: specifically enriching ectopic synthesis of blood coagulation factor X in tumor cells to obtain a protein sample; performing protein sample fixation and treatment on the protein sample by using SDS-PAGE gel electrophoresis, placing the gel in an ice box after scanning the gel by using a gel imaging system, and storing the gel; performing in-gel enzymatic digestion on the blood coagulation factor X in the gel to obtain a peptide segment solution; detecting the endogenous synthesis of blood coagulation factor X in tumor cells by using mass spectrometry technology, and obtaining mass spectrometry data. The application firstly adopts the mass spectrometry technology to detect the blood coagulation factor X synthesized by tumor cells outside normal liver cells, specifically enriches the ectopic synthesis of blood coagulation factor X in glioma cells by using the protein immunoprecipitation technology, performs SDS-PAGE gel electrophoresis, performs in-gel enzymatic digestion, and detects the endogenous synthesis of blood coagulation factor X in tumor cells by using the mass spectrometry technology.

This invention discloses an injury-related endogenous polypeptide and its applications. The invention employs a "single-model initial screening – multi-model cross-validation" strategy, using an acute lung injury (ALI) model as the screening set and partial hepatectomy (PHx) and cerebral ischemia-reperfusion injury (tMCAO) models as the validation set. Combined with liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology, a group of serum endogenous polypeptides was screened. These polypeptides are novel epitope fragments generated by specific enzymatic digestion of precursor proteins such as fibrinogen α-chain and hemoglobin α / β subunits in the tissue injury microenvironment. Their expression levels are extremely low in healthy serum but exhibit explosive upregulation in injured states. Experiments have confirmed that this group of polypeptides shows significant responses in acute lung injury, liver injury, and cerebral ischemia-reperfusion injury models, and can serve as broad-spectrum biomarkers for multi-tissue injury, providing a novel detection target for the early diagnosis of multi-tissue injury in acute and critical clinical settings.

The invention belongs to the technical field of cosmetics, and particularly relates to a preparation method and application of a rumex hanus micromolecule polypeptide and amino acid solution. According to the method, protein contained in rumex hanus is subjected to step-by-step enzymolysis, and a specific peptide fragment is obtained by controlling the pH value and the enzyme type, so that efficient enzymolysis is realized, the purity of a product is improved, and a foundation is laid for application of the rumex hanus to the field of cosmetics. According to the method, through enzymolysis in different stages, the safe and reliable production principle is achieved, the yield of rumex hanus micromolecule polypeptide is increased, and the technical problem that cosmetic protein raw materials are difficult to transdermal is solved.

The invention relates to the technical field of stem cell engineering and anti-aging biology, in particular to a histone modification regulation method based on placenta amnion sub-totipotent stem cells and an anti-aging preparation thereof. The regulation and control method comprises the following steps: separating and purifying healthy full-month caesarean section placenta amniotic membrane tissues, carrying out composite enzymolysis and flow cytometry screening to obtain CD105 < + >, CD73 < + > and CD45 <-> stem cells, and primarily culturing the CD105 < + >, CD73 < + > and CD45 <-> stem cells; the method comprises the following steps: determining histone methyltransferase MLL3 and demethylase JMJD3 as target spots through ChIP-seq, and determining H3K4me3 and H3K27me3 as target modification sites; an MLL3 activator CM-272 with the final concentration of 15 [mu] M and a JMJD3 inhibitor GSK-J4 with the final concentration of 10 [mu] M form a composite regulator, and induction culture is performed for 84 h under the conditions of 37 DEG C and 5% CO2; and carrying out Western Blot verification and screening on the stem cells meeting the standard. According to the anti-aging preparation, regulated stem cells are used as active ingredients, a sodium alginate-gelatin (2: 1) composite carrier, 5% mannitol and 1% human serum albumin protective agent are used as auxiliary materials, and the injection with the pH of 7.2-7.4 is prepared. The method is accurate in regulation and control, and the preparation is remarkable in anti-aging effect and low in immunological rejection risk.

This invention discloses a magnetic bead-based low-abundance protein enrichment kit and its extraction and enzymatic digestion method, relating to the field of biomedical technology. The kit includes a lysis buffer, magnetic bead washing buffer, reducing alkylation solution, two equilibration solutions, and a termination reagent. The extraction and enzymatic digestion method comprises the following steps: S1, low-abundance protein enrichment; S2, proteolytic digestion. This magnetic bead-based low-abundance protein enrichment kit and its extraction and enzymatic digestion method can rapidly and efficiently capture low-abundance proteins from bodily fluid samples, solving the problems of low digestion efficiency and frequent failures. Protein extraction is performed using an enzyme-compatible buffer, allowing the protein to be digested in the buffer, simplifying the operation. In particular, the application of multifunctional magnetic beads not only improves the enrichment efficiency of low-abundance proteins but also enhances the flexibility and throughput of analysis, opening up new prospects for proteomics research.

This invention discloses a method for the fusion of *Hymenochloa crus-galli* and *Phragmites australis* bioplasts and their callus regeneration, comprising the following steps: Step 1, callus suspension cell culture: Granular callus is induced using young spikelets of *Hymenochloa crus-galli* and *Phragmites australis* to establish a callus suspension cell line; Step 2, protoplast isolation and purification: Protoplasts are isolated using enzymatic digestion and purified using filtration-centrifugation; Step 3, protoplast fusion and callus regeneration: *Hymenochloa crus-galli* and *Phragmites australis* bioplasts are fused using an electrofusion method; the fused protoplasts are then cultured at 27-28°C in the dark / diffuse light environment to obtain regenerated callus from the fused protoplasts. The steps involved in this invention form a complete culture system, with each step closely connected to form a whole, laying the foundation for obtaining interspecific protoplast fusion and regeneration of plants within the *Phragmites australis* genus.

The invention provides a method for separating and extracting amniotic mesenchymal stem cells from a placenta. The method comprises the following steps: S1, pretreating the placenta; s2, amniotic membrane tissue separation; s3, tissue crushing; s4, enzymolysis digestion: softening the fragments, and mixing the softened fragments with a compound enzyme solution according to a mass-volume ratio of 1: (8-12) to obtain an enzymolysis product; s5, centrifugal separation: performing gradient centrifugation on the enzymolysis product to obtain cell precipitate; s6, cell culture: inoculating cell precipitates obtained by centrifugation to a compound culture medium; and step S7, immune sorting: separating target cells by adopting CD90 antibody coated magnetic beads. According to the method disclosed by the invention, three defects of cell injury, insufficient purity and low yield are synchronously overcome through a softening liquid protection mechanism, multi-enzyme synergistic dissociation, gradient centrifugal layering and other core technologies; and standardized parameter control (such as thickness cutting and temperature gradient) ensures the process reproducibility, and provides high-activity and high-purity functional stem cells for clinical application.