110 results about "Immunoprecipitation" patented technology

The invention relates to the technical field of plant genetic engineering, in particular to an application of an alfalfa MsPL19 protein and a gene thereof in regulating and controlling the plant height, fresh weight or biological yield of alfalfa. The invention provides an MsPL19 protein, a gene and application thereof, which can obviously improve the plant height, the biological yield and the fresh weight of medicago sativa and further improve the quality of the medicago sativa. Meanwhile, it is found that the MsPL19 protein and gene can respond to low temperature, drought, salt, hormone and other pathways, the protein interacting with MsPL19 is obtained through immunoprecipitation-mass spectrometry analysis, corresponding molecular modules can be enriched, the molecular mechanism of alfalfa MsPL19 responding to the low-temperature environment is analyzed, new cold-resistant varieties can be bred for alpine regions, and the application prospect is broad. And a new breeding direction is provided for increasing the yield of alfalfa.

The invention belongs to the technical field of antibody preparation, and particularly relates to an anti-human carbohydrate antigen CA125 monoclonal antibody and application thereof. The amino acid sequences of CDR1-3 on a light chain variable region of the monoclonal antibody are respectively shown as SEQ ID NO.3-5, and the amino acid sequences of CDR1-3 on a heavy chain variable region of the monoclonal antibody are respectively shown as SEQ ID NO.8-10. The monoclonal antibody provided by the invention can specifically recognize and bind to a natural carbohydrate antigen CA125 expressed by human cells or tissues, has good affinity and relatively high sensitivity of binding of the antibody and the antigen, has strong anti-interference ability to complex non-target protein components in the cells / tissues, is beneficial to significantly improving the accuracy and reliability of immunodetection of human CA125 protein, and has good application prospects. The method is suitable for high-specificity, high-sensitivity and high-reliability detection of the human CA125 protein, and has good applicability in a plurality of detection systems such as immunoblotting, immunoprecipitation, immunohistochemistry and the like.

The invention relates to application of an ACBD3 inhibitor in preparation of a medicine for treating lung cancer and gRNA for inhibiting ACBD3 gene expression, and belongs to the field of bioengineering.199 binding proteins of ACBD3 are obtained through co-immunoprecipitation and mass spectrum identification, new biological functions of ACBD3 are excavated through GO analysis and KEGG pathway analysis of the binding proteins of ACBD3, and the application of the ACBD3 inhibitor in preparation of the medicine for treating lung cancer is developed. The ACBD3 is used for regulating and controlling a KEGG pathway enrichment map of a differential gene, and the ACBD3 is found to participate in the occurrence and development of cancers; the ACBD3 is successfully knocked out by designing gRNA, and tests prove that the multiple effects of slowing down A549 lung cancer cell proliferation, inhibiting lung cancer cell migration, inhibiting A549 lung cancer cell invasion, retarding A549 cell cycle G1 phase and reducing tumorigenicity can be achieved by knocking out the ACBD3.

The present disclosure relates to methods of identifying RNA targets of RNA binding proteins. In aspects, the disclosure relates to a method of identifying RNA molecules bound by RNA binding proteins. Some embodiments of the present disclosure relate to a method that can definitively identify direct RNA-target interactions with targeted proteins without the requirement for immunoprecipitation or gel extraction. In some embodiments, the method may include combining multiple antibodies in the same sample.

The invention discloses an anti-soluble transferrin receptor monoclonal antibody as well as a preparation method and application thereof, and belongs to the technical field of antibody detection. According to the antibody, an L101-F760 amino acid sequence of human TfR protein is used as an immunogen, specific screening of B cells, reverse RNA (Ribonucleic Acid) into cDNA (Complementary Deoxyribose Nucleic Acid) through RT-PCR (Reverse Transcription-Polymerase Chain Reaction), then homologous recombination is performed to construct heavy and light chain vectors, and finally multiple rounds of screening are performed to obtain the purified sTfR monoclonal antibody, so that the sTfR protein can be specifically recognized, and the antibody has the advantages of high specificity and high sensitivity and can be widely applied to the field of immunotherapy. The natural sTfR protein in human serum can be recognized. The kit is applied to the detection and screening fields of immunohistochemistry, immunofluorescence chemistry, western blot, co-immunoprecipitation, indirect ELISA, flow cytometry and the like, especially has low background and no interference of non-specific binding impure protein during immunohistochemistry detection, can obtain more accurate detection and evaluation results, avoids interference of false positive and false negative, and has high detection accuracy. The method has great application value in the aspects of basic scientific research and clinical pathology detection.

The invention discloses a high-flux and rapid chromatin immunoprecipitation sequencing method based on a HiBiT tag and a Tn5 enzyme, and belongs to the technical field of biology. According to the method, the high affinity characteristic of a HiBiT tag system is combined with a Tn5 enzyme in-situ library building technology, firstly, a DNA binding protein recombinant expression strain containing a HiBiT tag is constructed and subjected to induced expression, after formaldehyde crosslinking and cell lysis, an Anti-HiBiT antibody is used for rapid immunoprecipitation, and a Tn5 enzyme library is constructed; tn5 transposase is directly loaded on the magnetic beads combined with the protein and DNA fragment compound to complete fragmentation and linker connection, so that the construction of a next-generation sequencing library is quickly carried out, and the total consumed time from immunoprecipitation to the completion of library construction is less than or equal to 5 hours. Meanwhile, automatic treatment is realized through a micro experiment board (the reaction system is less than or equal to 200 microliters) and a liquid workstation, the experiment cost and the sample dosage are reduced, 48 transcription factors and 48 control samples can be synchronously treated, the efficiency is greatly improved, and an efficient tool is provided for microbial transcription regulation and control research.

The invention discloses an RNA fragment extraction method, and relates to the technical field of molecular biology. Comprising the following steps: S1, preparing materials; s2, cell culture: S21, cell resuscitation; s22, performing cell cryopreservation; s23, carrying out cell passage; s3, co-immunoprecipitation, specifically comprising the following steps: S31, binding an antibody to a protein A / G magnetic bead; s32, carrying out cross-linking combination on the antibody; s33, carrying out cell lysis treatment; s34, carrying out manual antigen immunoprecipitation; s4, carrying out RNA extraction on the basis of a TRIZOL method; and S5, carrying out reverse transcription and real-time fluorescent quantitative PCR. By improving the components of the lysis solution and the washing solution, the extraction efficiency and the yield of RNA are improved, and meanwhile, the pollution of impurities is reduced; the usage amount of reagents and experiment steps are reduced, and the experiment cost is reduced.

The invention relates to a method for screening tumor autoantibodies by using a small peptide display library. The method comprises the following steps: S1, obtaining a tumor mutation small peptide display library; s2, screening to obtain small peptides capable of interacting with serum; s3, analyzing protein and mutation information in the tumor mutation material to obtain an analysis result; and S4, calculating to obtain protein and mutation information with significant difference. Therefore, on the basis of obtaining a tumor mutation material (related), serum immunoprecipitation and high-throughput sequencing are combined, and a strategy for globally and unbiasedly researching the mutation-related tumor autoantibody is provided.

The invention discloses a methionine nutrition selection marker saccharomyces cerevisiae vector as well as a construction method and application thereof, and relates to the field of genetic engineering and synthetic biology, a methionine MET17 promoter and a coding region sequence are obtained through gene artificial synthesis, a leucine promoter and a coding region sequence in a pAG415-GAL1-ccdB vector are replaced by homologous recombination, and thus the methionine nutrition selection marker saccharomyces cerevisiae vector is obtained. A pAG415-GAL1-MET-ccdB expression vector is constructed and obtained, a ccdB gene is replaced by a green fluorescent protein reporter gene GFP sequence to be integrated into the pAG415-GAL1-MET-ccdB vector, a pAG415-GAL1-GFP-MET vector is constructed and obtained, the obtained expression vector can be used for screening exogenous gene transformants in a methionine deficient culture medium, meanwhile, green fluorescent GFP tag protein carried in the vector has the advantages that the expression vector can be applied to screening of the methionine deficient culture medium, and the expression efficiency of the methionine deficient culture medium is improved. And more application space is provided for carrying out subcellular localization, immunoprecipitation and other downstream inheritance and molecular biology analysis on a target gene in wine brewing cells.

The invention discloses a whole genome methylation detection method based on MeDIP-MSRE, and belongs to the technical field of epigenetics detection. According to the method, methylation immunoprecipitation sequencing and a methylation sensitive restriction enzyme technology are innovatively combined, firstly, a methylation specific antibody is used for conducting immunoprecipitation on sample DNA, and whole genome methylation fragments are enriched; then carrying out enzyme digestion on the enriched product by adopting methylation sensitive restriction enzyme, specifically removing an unmethylated DNA region, and reserving a complete methylation sequence; and finally, constructing a methylation map through high-throughput sequencing. According to the method, traditional hydrosulfite chemical conversion is not needed, DNA damage and base conversion deviation caused by the traditional hydrosulfite chemical conversion are avoided, meanwhile, high sensitivity of MeDIP and high specificity of methylation sensitive restriction endonuclease are fused, and the fidelity, sensitivity and specificity of detection are remarkably improved.

The application belongs to the technical field of biology and particularly relates to an African swine fever virus pNP868R protein monoclonal antibody and a preparation method and application thereof. The application takes the pNP868R protein prepared by eukaryotic expression as an antigen, immunizes BALB / c mice, uses hybridoma technology, and obtains a hybridoma cell line capable of stably secreting the anti-African swine fever virus pNP868R protein monoclonal antibody through cell fusion, indirect ELISA method screening, limited dilution method subcloning and hybridoma continuous passage culture. The application also obtains an African swine fever virus pNP868R protein monoclonal antibody. The monoclonal antibody has high affinity with the pNP868R protein and can be applied in Western-Blot experiments, indirect immunofluorescence experiments, immunoprecipitation experiments and the capture of proteins interacting with pNP868R, and can be used for the diagnosis, prevention or treatment of African swine fever.

The invention relates to the technical field of biological medicines, in particular to application of panthenol-cytochrome c reductase core protein 2 lactic acid modification as a therapeutic target in preparation of drugs for treating sepsis cardiomyopathy. The invention discloses a key effect of Uqcrc2 K109 site lactic acid modification in SIC and application of the Uqcrc2 K109 site lactic acid modification as a therapeutic target. The Uqcrc2-K109 is found to be a therapeutic target of the SIC through molecular biology methods such as proteomics analysis, lactylation omics analysis and co-immunoprecipitation, and the function of a mitochondrial respiratory chain complex III is recovered in a targeted manner by regulating and controlling the lactylation level of the Uqcrc2-K109, so that a new strategy is provided for treatment of the SIC.

The invention belongs to the technical field of biological medicines, and relates to a Rubicon permeable membrane peptide and application thereof. Specifically, the permeable membrane peptide fragment sequence is designed on the basis of an amino acid sequence of interaction of Rubi protein and Btk, and seven permeable membrane sections of arginine are connected to the tail end of the permeable membrane peptide fragment sequence through disulfide bonds. Immunofluorescence experiments prove that the peptide fragment can efficiently penetrate a platelet membrane to enter cells, and co-immunoprecipitation experiments prove that the peptide fragment has strong affinity with Btk. Platelet aggregation experiments show that the peptide fragment can significantly inhibit platelet activation under collagen stimulation. In an animal model, the peptide fragment is injected before instantaneous middle cerebral artery occlusion or reperfusion of a mouse, and ischemia-reperfusion injury can be effectively relieved. The invention provides a novel potential drug candidate for treating thrombotic diseases and ischemia-reperfusion injury.

The invention relates to methylation markers of colorectal polyp / adenoma and colorectal cancer as well as a screening method and application of the methylation markers, and further relates to a construction method of a three-classification model for simultaneously screening colorectal polyp / adenoma and colorectal cancer. Related methylation markers are screened by adopting a methylation co-immunoprecipitation sequencing technology, and a multi-stage classification model is constructed through a characteristic methylation difference region analysis algorithm, so that classification prediction of normal healthy people, colorectal polyp / adenoma people and colorectal cancer people can be realized, high sensitivity is achieved, precancerous lesions can be found as early as possible, and the clinical application prospect is wide. And a new technical scheme is provided for early screening of colorectal cancer.

The present invention belongs to the technical field of sensorineural hearing. Provided is the use of SPTBN1 as a target in the treatment of GJB2-related sensorineural hearing loss. The use comprises: the construction of a stably transfected cell line, wherein: HEK293T cells are cultured using a DMEM culture medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified incubator at 37ºC with 95% air and 5% CO2, and when the cells reach 80%-90% confluence, cell passage is performed; IP-MS analysis, wherein: protein complexes are purified using Protein A / G immunoprecipitation magnetic beads, a portion of the extracted proteins is used as input, and then 2 μg of anti-Cx26 antibody is added to the remaining protein extract, same are gently pipetted and mixed, and incubated on a rotating shaker at 4℃ overnight; immunofluorescence observation of the co-localization of Cx26 and SPTBN1, wherein: a stably transfected cell line expressing WT-Cx26 and Mut-Cx26 is constructed; and co-immunoprecipitation (Co-IP) validation, wherein: a stably transfected cell line expressing WT-Cx26 is constructed. The present invention overcomes the limitations of therapeutic methods, such as the relatively low targeting specificity and short therapeutic time windows associated with full-length protein supplementation via gene therapy, thereby providing new insight into GJB2-related hearing loss, and a new target and a new treatment for same.

The present invention is directed to the field of in vitro diagnostics and relates to reagents containing non-blood-based antibodies for determining an analyte in an immunoprecipitation assay and having a sensitivity comparable to conventional reagents containing polyclonal antibody sera.

The invention relates to a biotinylation modified amino acid probe, a preparation method and application thereof, and a method and application for identifying amino acid binding protein. The amino acid probe is amino acid with biotin modification at an amino terminal or a carboxyl terminal; or, the amino acid probe is amino acid with an azide group at the carboxyl end, the amino acid probe reacts with alkynyl-containing biotin under the catalysis of copper ions through a click chemical reaction in a cell to generate biotin-modified amino acid, an azide-modified amino acid probe library is supplemented, and the amino acid probe is combined with immunoprecipitation and mass spectrometry technologies, so that the biotin-modified amino acid is obtained. Proteins capable of being directly combined with amino acids can be identified, an important tool is provided for follow-up research of amino acid sensing proteins, and the method has important application value in identification of amino acid binding proteins, research of amino acid sensing mechanisms and functions and determination of drug targets or screening of lead compounds.

The invention provides a phosphorylated protein enrichment method, and relates to the fields of materials, analytical chemistry and life science. The enrichment method of the phosphorylated protein comprises the following steps: dissolving a phosphorylated protein sample in a sample loading solution, mixing with super-hydrophilic metal ion functionalized microspheres, hatching, centrifuging, and discarding supernatant to obtain a super-hydrophilic material enriched with the phosphorylated protein; mixing the material enriched with the phosphorylated protein with an elution solution, incubating, and centrifuging to obtain enriched liquid of the phosphorylated protein; the super-hydrophilic metal ion functionalized microsphere is provided with a super-hydrophilic nano convex shell which is completely coated on the solid core. The enrichment method provided by the invention can overcome the non-specific adsorption problem caused by modification defects of a traditional monomolecular layer modified functional material, and has excellent enrichment selectivity; the method can overcome the limitation of an immunoprecipitation enrichment method antibody, has wide applicability, and is hopeful to occupy an important position in phosphorylated protein separation and enrichment.

The present application relates to the technical field of plant genetic engineering, and particularly relates to an application of Medicago sativa MsPL19 protein and gene in regulating plant height, fresh weight or biological yield of Medicago sativa. The present application provides the MsPL19 protein, gene and application thereof, which can significantly improve the plant height, biological yield and fresh weight of Medicago sativa, and further improve the quality of Medicago sativa. Meanwhile, it is found that the MsPL19 protein and gene can respond to low temperature, drought, salt, hormone and other pathways, the proteins interacting with the MsPL19 are obtained through immunoprecipitation-mass spectrometry analysis, the corresponding molecular module can be enriched, the molecular mechanism of Medicago sativa MsPL19 responding to low temperature environment is analyzed, and a new cold-resistant variety can be selected in an alpine region, thereby providing a new breeding direction for improving the yield of Medicago sativa.

Methods of determining disease load or type in a subject suffering from a disease associated with cell death of a specific tissue or cell type are provided. Methods of determining a cell free DNA chromatin immunoprecipitation and sequencing (cfChIP-Seq) marker and methods of classifying a subject suffering from a disease are also provided.

Provided herein is a method of detecting an amyloid peptide in a patient sample, including exposing the patient sample to a binding reagent in the presence of an assay binding buffer, thereby immunoprecipitating the amyloid peptide; washing the immunoprecipitated amyloid peptide; eluting the washed, immunoprecipitated amyloid peptide, thereby generating free amyloid peptide; and analyzing the free amyloid peptide with a mass spectrometer.

The invention provides a photolysis group-containing antibody, a method and a kit for co-immunoprecipitation sequencing. The photolysis group-containing antibody comprises a first antibody and a linker reagent label, and the first antibody is subjected to photolysis-bifunctional labeling through a linker reagent; the linker reagent comprises three active groups R1, R2 and R3; wherein R1 is used for covalent linkage with a first antibody; r2 is a photolysis group and fractures under illumination of a preset wavelength, so that connection between R1 and R3 is broken; r3 is used for carrying out binding reaction with a solid-phase substrate; r1, R2 and R3 are connected through a length-adjustable flexible chain, and the length-adjustable flexible chain is of a polyethylene glycol or carbon chain structure; according to the method, interference of complex backgrounds in cell samples can be reduced, the sample input quantity is reduced, and low-abundance epigenetic modification is realized.

The invention belongs to the field of biological medicine, and particularly relates to PGK1 K6 site succinylation modification and application. Experiments show that the 6th lysine of the PGK1 protein has succinylation modification (PGK1 K6su), and the existence of the succinylation modification at the 6th lysine is confirmed through immunoprecipitation experiments; cell function experiments prove that the PGK1 K6su enhances lung cancer cell migration and multiplication capacity and promotes lung cancer progression. A small molecule compound diammonium glycyrrhizinate aiming at the PGK1 K6 site is further obtained through computer-assisted screening, and subsequent experiments find that the compound can inhibit proliferation and migration of lung cancer cells by reducing the succinylation modification level of the lung cancer cells PGK1 K6.

The invention discloses a method for inhibiting lipid metabolism of nasopharyngeal carcinoma (NPC) through an LncRNA ANRIL-ZBTB7A pathway. The invention relates to a method for inhibiting lipid metabolism of nasopharyngeal carcinoma by using an LncRNA ANRIL-ZBTB7A pathway, which comprises the following steps: carrying out modification treatment on LncRNA ANRIL so as to negatively regulate an SREBF1-FASN factor and further inhibit lipid metabolism of NPC; when the transformation treatment mode is that the LncRNA ANRIL is over-expressed, the ZBTB7A is knocked down. According to the application disclosed by the invention, the LncRNA ANRIL is combined with shZBTB7A and an SREBP inhibitor famostatin, so that the lipid metabolism of NPC is inhibited, and an important role is played in research and development of targeted therapeutic drugs. The independently designed primer sequences of the chromatin immunoprecipitation-quantitative polymerase chain reaction of the LncRNA ANRIL, the SREBF1 and the FASN can be used as a new way for researching the NPC.

The application provides a biomarker for early colorectal cancer detection and application thereof, uses an immunoprecipitation sequencing platform (mDIP-Seq) based on high-resolution mRNA display technology, screens a plasma antibody diagnostic marker of early colorectal cancer, combines a random forest algorithm to develop a multiple antibody panel suitable for early colorectal cancer diagnosis, constructs a disease prediction model, and improves the accuracy of early colorectal cancer diagnosis. Compared with invasive colonoscopy, the method has the characteristics of low cost, generalization and high throughput, and is suitable for large-scale early colorectal cancer screening in the population.

The invention relates to a fusion protein GLuc-p30 and a preparation method and application thereof. With p30 protein of African swine fever as a diagnostic antigen, an African swine fever antibody detection method based on a luciferase immunoprecipitation system is established. In the method, a fusion antigen of luciferase and p30 is prepared by DNA transfection of eukaryotic cells, the antigen is secreted outside the cell, the fusion antigen is combined with African swine fever antibodies in a serum sample, and then captured by magnetic beads coupled with Staphylococcus aureus protein A, and the fusion antigen specifically combined with the magnetic beads is detected by a luciferase test; the virus antigen expressed by the eukaryotic cells used in the invention contains post-translational modifications such as phosphorylation, is closer to the virus protein expressed under infection, and does not need to be purified, thereby simplifying the preparation process of the antigen; compared with the existing indirect ELISA, the detection method of the invention has better detection sensitivity and specificity; the detection method of the invention has the advantages of simplicity, sensitivity, specificity, low price, etc., and can meet the requirements of large-scale detection.

In the analysis method according to the present invention, a predetermined peptide is separated by immunoprecipitation using an antibody which specifically binds to either an N-terminus or a C-terminus of the predetermined peptide. The separated predetermined peptide is digested with a protease to prepare peptide fragments, and among the peptide fragments, a peptide fragment at a terminus opposite to a terminus binding to the antibody is mass-spectrometrically detected.

This invention provides a sugarcane ShERF1b Genes, proteins, and applications belong to the field of biotechnology, and specifically disclose sugarcane... ShERF1b Application of genes in enhancing sugar accumulation. This invention screens genes that are specifically upregulated in mature sugarcane stem nodes and are potential key regulatory transcription factors controlling sugar accumulation. ShERF1b And cloned cultivated sugarcane ShERF1b The full-length gene was constructed, and overexpression and gene editing vectors were constructed. These vectors were then genetically transformed into cultivated sugarcane to elucidate the gene function regulating sugar accumulation in sugarcane. Simultaneously, downstream target genes were isolated using immunoprecipitation to analyze the molecular pathways and networks regulating sugar accumulation in sugarcane, thus providing a preliminary understanding of the gene's role in this process. ShERF1b The molecular mechanism regulating sugar accumulation in sugarcane provides a theoretical basis and genetic resources for the improvement of high-sugar sugarcane varieties.

The invention provides application of SPTBN1 as a target spot in treatment of sensorineural hearing loss related to GJB2, and belongs to the technical field of sensorineural hearing. Comprising the following steps: constructing a stably transfected cell line: culturing HEK293T cells in a humidifying incubator with temperature of 37 DEG C, 95% of air and 5% of CO2 by using a DMEM (Dulbecco Modified Eagle Medium) culture medium added with 10% of fetal calf serum and 1% of double antibodies, and performing cell passage when the cells grow to a confluence degree of 80-90%; performing IP-MS analysis: purifying a protein compound by using ProteinA / G immunoprecipitation magnetic beads, taking part of extracted protein as input, then adding 2 [mu] ganti-Cx26 antibody into the residual protein extract, slightly blowing, beating and mixing, and performing turnover shaking table incubation at 4 DEG C overnight; carrying out immunofluorescence observation on co-localization of the Cx26 and the SPTBN1; constructing a stably transfected cell line for expressing the WT-Cx26 and the Mut-Cx26; and carrying out co-immunoprecipitation (Co-IP) verification: constructing a stably transfected cell line for expressing the WT-Cx26. According to the invention, limitations of treatment modes such as relatively low complete protein targeting and short treatment time window are supplemented through gene therapy, and a new insight, a new target spot and a new treatment are provided for GJB2-related hearing loss.

This disclosure relates to the field of mass spectrometry analysis. In some embodiments, the disclosure relates to compositions and methods for detecting and quantifying proteins in the AKT-mTOR pathway by immunoprecipitation enrichment followed by mass spectrometry analysis.