36 results about "Immunoprecipitation" patented technology

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 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 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.

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 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.

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.

The application discloses a stapling peptide targeting TRIB3 / SSRP1 interaction and application of the stapling peptide in preparation of anti-multiple myeloma drugs. Through a series of experiments such as immunoprecipitation coupling mass spectrometry, it is found that TRIB3 directly interacts with SSRP1 protein in multiple myeloma cells, thereby playing a role in promoting malignant proliferation of multiple myeloma, and TRIB3 promotes degradation of the SSRP1 protein through a ubiquitin-proteasome pathway. The stapling peptide 7695-SP5 inhibits malignant proliferation of multiple myeloma by specifically inhibiting TRIB3 / SSRP1 protein interaction. The stapling peptide 7695-SP5 can be used as a TRIB3 / SSRP1 protein interaction inhibitor to treat diseases caused by increased expression of TRIB3 and SSRP1 proteins, such as multiple myeloma, and is expected to be developed into a novel anti-tumor drug.

The invention discloses a cervical cancer influence mechanism research method based on ICAT regulated ferroptosis, and relates to the technical field of molecular biology. Comprising the following steps: evaluating the ferroptosis effect of ICAT in cervical cancer; the ICAT is verified to promote the malignant progression of cervical cancer by inhibiting ferroptosis; the molecular mechanism of ICAT for inhibiting cervical cancer ferroptosis is clarified; and screening an anti-cervical cancer candidate compound. According to the invention, the action downstream target of ICAT is accurately positioned as GPX4 through proteomics and immunoprecipitation-mass spectrometry, and the specific mechanism of stabilizing GPX4 protein by influencing ZDHHC5-mediated palmitoylation is clarified, so that the target is very clear; a targeted drug screening strategy is designed for screening small molecule compounds, antibodies or nucleic acid drugs capable of targeting ICAT or destroying GPX4 palmitoylation, and a clear target spot and a feasible screening path are provided for developing novel anti-cervical cancer drugs.

The application discloses an application of an ND-42 related plasmid in preparation of a Drosophila mitochondrial protein expression kit, and the kit is mainly designed for a preparation method of mitochondrial related protein expression in S2 cells for the ND-42 protein. The kit constructs a pUAS-attB-3xHA-ND-42CDS full-length plasmid, a pUAS-attB-3xHA-ND-42-DNK-dom plasmid and a pUAS-attB-3xFlag-Nap1CDS full-length plasmid. The kit mainly uses a UAS-Gal4 system to realize the expression of the ND-42 protein, the DNK domain of the ND-42 protein and the Nap1 protein. The results of immunoprecipitation and Western blotting show that the Nap1 protein can strongly interact with the ND-42 protein. Further experimental observation shows that the Nap1 protein can also strongly physically combine with the DNK domain of the ND-42 protein. The kit can provide an important research method for the function and mechanism exploration of the mitochondrial related protein.

The invention relates to the field of biological medicine and immunotherapy, and particularly provides a liver cancer neoantigen vaccine based on immunopeptidomics and immunotherapy application of the liver cancer neoantigen vaccine. According to the invention, an MHC-I immunoprecipitation technology is combined with high-throughput mass spectrometry, a neoantigen short peptide group with excellent immunogenicity is identified from a liver cancer sample, and a long peptide group is obtained by extension on the basis of short peptides. The oligopeptide antigen and the long peptide antigen are respectively mixed with an adjuvant Poly (I: C) to prepare a vaccine, and a mouse liver cancer model proves that the vaccine can effectively inhibit tumor progression and induce a tumor microenvironment to be converted into an immune activation state. The invention provides a new antigen source and a vaccine form for individualized liver cancer immunotherapy, and has remarkable clinical application potential and industrialization prospect.

The invention discloses an RNA co-immunoprecipitation kit for specific binding of prion protein in muscle cells and an RNA extraction method, and relates to the technical field of bioengineering. According to the present invention, the specific co-precipitation kit preparation and the specific extraction steps comprise cell lysis, cell lysis solution-antibody-magnetic bead incubation, protein digestion and RNA extraction, such that the high purity and the sufficient amount of the enriched RNA are successfully ensured so as to completely meet the strict requirements of the subsequent RNA-seq sequencing. The method overcomes the inherent defects of the existing RIP technology in the research of non-classical RNA binding proteins, realizes the optimization of the whole process from high-specificity enrichment to high-quality sequencing sample preparation through the integrated innovation of methodology and the kit, has the outstanding advantages of strong specificity, high sensitivity, good repeatability, simple operation and the like, and has a wide application prospect in the research of non-classical RNA binding proteins. And remarkable technical progress is achieved, and a positive application effect is generated.

This invention relates to the field of biotechnology, specifically to a method for elucidating the interaction complex between the BTVNS2 protein and host cells. The method involves constructing an initial list of host proteins through cross-validation using affinity purification-mass spectrometry and biotinylate proximity labeling-mass spectrometry. Weighted scoring and ranking are then performed based on quantitative mass spectrometry data and functional relevance. Binding characteristics are validated and quantified intracellularly and extracellularly using immunoprecipitation and biophysical techniques, respectively. The complex is assembled in vitro, and its high-resolution three-dimensional structure is resolved using single-particle cryo-electron microscopy. This invention addresses key problems in existing technologies, such as incomplete capture of interacting proteins, high false-positive rates, blind functional screening, and difficulty in obtaining samples suitable for high-resolution structural analysis due to reliance on single methods. It systematically identifies key host factors and ultimately reveals the precise structure of the interaction interface at the atomic level, providing a foundation for understanding viral replication mechanisms and developing antiviral strategies.

The present invention reveals MESD as a critical host dependency factor for Oropouche virus (OROV) infection. Initial identification was achieved through a CRISPR-Cas9 loss-of-function screen, which highlighted MESD amongst other potential factors. The ensuing research demonstrated that MESD knockout in HEK293T cells resulted in a significant reduction in OROV infection rates. Complementing MESDKO cells with FLAG-MESD re-established susceptibility, thus confirming MESD's essential role in the infection process. Interestingly, MESD specifically facilitates OROV infection without enhancing the infectivity of other viruses such as LACV, TOSCV, and WNV. The specificity of MESD for OROV was further validated by its interaction with OROV envelope glycoproteins, as shown through immunoprecipitation assays. These findings suggest that targeting MESD could be a novel and effective approach for diagnosing and treating OROV infections, potentially paving the way for innovative therapeutic strategies against this virus.

Disclosed in the present invention is the use of an anti-Gelsolin3 antibody related to demyelinating Guillain-Barré syndrome. The use mainly comprises the following steps: collecting the serum from a GBS patient and a control individual, performing co-immunoprecipitation with a sciatic nerve protein to obtain an immune complex, separating and identifying the immune complex by using a mass spectrometer to obtain a candidate protein sequence, inserting a candidate protein coding gene into a vector, transfecting cells followed by immunofluorescence verification to determine the localization and expression of the candidate protein, constructing a candidate protein expression plasmid, expressing and purifying a recombinant protein in Escherichia coli, performing co-immunoprecipitation verification on the purified recombinant protein by using the serum from the GBS patient to determine the presence of an anti-Gelsolin3 IgG antibody, further verifying the expression of the anti-Gelsolin3 IgG antibody in the serum from the GBS patient by using a Western blot method, and verifying the binding of the serum from an anti-Gelsolin3 IgG antibody-positive patient to the sciatic nerve by using a single-fiber immunofluorescence method.

The invention discloses a small-scale TELP-assisted rapid chromatin immunoprecipitation sequencing method. MNase digestion is adopted to replace ultrasonication, so that the problem of epitope masking possibly caused by cross-linking is avoided. Library construction is carried out by using a TELP method, and efficient capture is realized by using an anchor primer tailed by PolyC and labeled by biotin. Only 3 '-phosphoric acid is removed from the rSAP, preparation is made for a subsequent tailing reaction, thermal inactivation can be achieved, the purification step is omitted, a traditional terminal repair enzyme system is not needed, and TELP has high sensitivity to low-initial-quantity DNA and is suitable for trace DNA produced by ChIP-seq. And through streptavidin magnetic bead capture, all subsequent steps (washing and connecting) are performed on the magnetic beads, so that the sample transfer loss is greatly reduced. 3 '-OH is directly used for tailing, and the steps of tail end repairing and A tail adding which are most likely to cause loss in traditional library building are omitted. According to the method, histone modification can be detected only by using hundreds of cells.

The invention belongs to the technical field of protein extraction, and particularly relates to an extraction method of AQP4 protein. 4% OGP is adopted for mild dissolution, 5% glycerin is used for antigen protection, the activity retention rate is larger than or equal to 85%, the activity retention rate is only about 50% due to denaturation of a glycine acid eluent in an immunoprecipitation method, and the method is more suitable for functional and immune experiments. The method is a high immunocompetence extraction method aiming at AQP4, the purity of the purified AQP4 is larger than or equal to 80%, the autoimmunocompetence of the antigen is sufficiently reserved, and the requirements of structural analysis, functional experiments and reagent development are met. The method is low in cost, high in efficiency, low in equipment dependence degree and suitable for large-scale preparation, and high-valence antibodies are prevented from being used, and an ultra-speed centrifuge is not needed.

The invention discloses an application of an IGF2BP1 fragment in preparation of an inhibition tool for screening and blocking the interaction between IGF2BP1 and SOX2 protein, and the IGF2BP1 fragment is an amino acid sequence from 161st to 578th of the IGF2BP1, and is shown as SEQ ID NO.01. The invention also discloses an application of the IGF2BP1 fragment in preparation of an inhibition tool for screening and blocking the interaction between IGF2BP1 and SOX2 protein. According to the present invention, the 161-578 amino acid fragment (IGF2BP1 [delta] C (161-578)) of the IGF2BP1 protein is the minimum functional structure domain of the IGF2BP1 protein directly interacting with the SOX2 protein, and the fragment sequence provides the clear targeting site for the subsequent accurate blocking of the IGF2BP1-SOX2 interaction through the dual verification of the bimolecular fluorescence complementation and the co-immunoprecipitation;

The invention discloses a library building method for detecting G-quadruplex structures in oocytes, early embryos and trace cells, and belongs to the technical field of whole genome immunoprecipitation library building. The DNA G-quadruplex structure provided by the invention is a DNA secondary structure which is different from a classic double-helix structure; g-quadruplex structure abnormity can damage telomere stability, genome stability, transcriptional activity and the like; this is an important reason for poor ovum quality and embryonic development retardation. The method provided by the invention can be used for evaluating the quality of the ova of the infertile women and judging reasons causing immaturity of the ova; abnormal enrichment of in-vivo and in-vitro development arrest embryo whole genome G-quadruplex is detected and can be used as an important index for screening reasons causing embryonic development retardation; the method can be used for guiding whether corresponding clinical patients are suitable for a series of assisted reproductive technologies (ART) such as in vitro fertilization-embryo transfer (IVF-ET) and the like.

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.

The present invention provides a method for determining the resistance of a microorganism to a drug by detecting at least a transcript of a drug resistance gene from a microorganism in a biological sample, the method comprising the steps of: (i) lysing the cells by means of a chemical or a mechanical method, thereby obtaining a lysate and cell debris; (ii) obtaining a ribosome-antibody complex from the lysate using an antibody or a fragment thereof which binds specifically to a microorganism-ribosomal protein; (iii) purifying the mRNA associated to the ribosome-antibody complex by means of a nucleic acid extraction method; and (iv) submitting the resulting mRNA to a specific gene detection method, thereby identifying the at least one drug resistance gene transcript of the biological sample. Provided methods and kits allow determining the resistance to antibiotics of a biological sample in a rapid and reliable manner, thereby minimizing the risk of AMR and allowing the definition of the therapeutic potential of a selected antibiotic

The application provides a pathogen immune spectrum evaluation method based on high-throughput antibody epitope spectrum detection data, comprising: data preparation: obtaining sequencing data of an antigen epitope library and sample sequencing data after immunoprecipitation of the biological sample and the library; data preprocessing: quality control of all sequencing data; data alignment and counting: constructing a reference library index, index alignment, and counting read counts of pathogen epitope peptides; identifying significantly enriched epitope peptides in the sample; calculating a calibration coefficient; calculating enrichment immune characterization features of the pathogen and determining whether it is a positive immune response. The EPIC score evaluation method of the application can efficiently process and summarize thousands of virus site information, accurately evaluate the immune state of an individual against multiple pathogens, extract key information from high-throughput detection data, accurately identify the binding of antibodies and antigen epitopes, and directly present the detection results in a visual image.

The invention discloses a detection method for detecting interaction strength of E1B-55K and p53 in cells, and relates to the technical field of biological detection. According to the method, a resolution type luciferase complementary technology is adopted, only several core steps of carrier construction, cell transfection, culture and fluorescence detection need to be completed, the operation process is simple, the whole detection process can be completed within 48-72 h, and compared with traditional methods such as co-immunoprecipitation, the detection time is remarkably shortened, and the detection efficiency is improved; the nano luciferase is used as a reporter molecule, has the characteristics of high luminous intensity, low background signal and high detection sensitivity, and can accurately detect the interaction between low-abundance proteins; meanwhile, a negative control and a positive control are set, and a relative fluorescence intensity value is calculated, so that non-specific interference can be effectively eliminated, and accurate quantification of the interaction intensity of E1B-55K and p53 is realized.

The application discloses a method for screening antiviral target points by targeting viral DNA, and effective sgRNA sequences targeting key DNA regions of varicella-zoster virus, herpes simplex virus type I and Kaposi's sarcoma-associated herpes virus are obtained respectively; dCas9-sgRNA containing viral DNA plasmid is co-transfected, and DNA of the varicella-zoster virus, the herpes simplex virus type I and the Kaposi's sarcoma-associated herpes virus is immunoprecipitated and enriched respectively to screen proteins regulating viral replication; the proteins are identified by mass spectrometry, and it is verified that the method is also applicable to screening proteins regulating viral replication by targeting HIV DNA, so that the method for screening proteins regulating viral replication by targeting viral DNA is established.

1. The name of the design product: immunoprecipitation device. 2. The use of the design product: the design product is a device for mixing immunoprecipitation samples. 3. The design points of the design product: in shape. 4. The picture or photo that best indicates the design points: perspective view.

The invention discloses an in-vivo protein-DNA interaction analysis method based on methylated DNA immunoprecipitation, and belongs to the technical field of molecular biology. According to the method, an instantaneous protein binding event is recorded as stable 6mA, MeIP captures interaction which cannot be detected by ChIP, and meanwhile, the sequence background limitation of DamID is avoided. The compatibility with qPCR and sequencing enables the experiment scale to be flexibly adjusted, and the simple implementation of the method enables the method to be very suitable for researching related proteins interacting with chromatin in organisms lacking endogenous DNA methylation. The invention also discloses the transient but critical localization of a kinase at the centromere, and emphasizes the necessity of capturing a dynamic chromatin event for understanding genome regulation. The MeIP technology can be applied to a mammalian cell system, and a powerful tool is provided for researching instantaneous chromatin regulation events in higher eukaryotes.

This invention provides a method for the enrichment and specific detection of extrachromosomal circular DNA (eccDNA) bound to target proteins, belonging to the field of gene-protein interaction technology. This invention provides a method for enriching eccDNA bound to target proteins. Based on the conventional chromatin immunoprecipitation procedure, it introduces a circular DNA enrichment step involving selective enzymatic digestion to remove linear DNA, achieving precise and highly specific enrichment of eccDNA bound to target proteins. This invention combines immunoprecipitation, selective enrichment of circular DNA, amplification, and high-throughput sequencing, and analyzes the results based on the identification of circular junctions, enabling genome-wide localization and systematic study of eccDNA bound to target proteins. This invention can accurately identify eccDNA bound to target proteins, providing important technical support for studying the role of eccDNA in gene expression regulation, chromatin conformation formation, and diseases such as tumors.