51 results about "Protein blotting" patented technology

Nanocrystals having a ZnTe core and methods for making and using them to construct core-shell nanocrystals are described. These core-shell nanocrystals are highly stable and provide quantum yields and stability suitable for applications such as flow cytometry, cellular imaging, and protein blotting, medical imaging, and other applications where cadmium toxicity is an issue.

The present invention relates to a membrane for Western blotting which has a three-dimensional open pore structure, an average pore diameter of 0.1-1.0 μm and a thickness of 30-200 μm, wherein the membrane for Western blotting is manufactured by subjecting nanofibers having an average fiber diameter of 50-1000 nm, obtained by electrospinning, to a hot-plate calendering process, and a method for manufacturing the same. The method comprises the steps of: dissolving a hydrophobic material in a solvent to prepare a spinning solution; subjecting the spinning solution to a spinning process to obtain a hydrophobic polymer nanofiber web; and calendering the obtained nanofiber web to obtain a membrane for Western blotting.

The present invention relates to a process for preparing anthrax protective antigen protein from E. coli using fed batch culture. This process creates a constitutively expressing system for rapid, efficient, cost-effective and high-level production of anthrax PA from E. coli. The steps of the process involves, transforming E. coli DH5α cells with the recombinant constitutive expression plasmid containing the PA gene to obtain recombinant DH5α cells and testing the PA expression by lysis of said cells followed by denaturing gel electrophoresis and Western Blotting technique using PA antibodies. This is followed by fermentation and harvesting of the high cell density cells. The said cells are solubilized using 6-8 Molar Urea and separated by centrifugation. The urea denatured PA is isolated from said supernatant and purified and thereafter eluted.

A preparation method of temperature sensitive magnetic western-blotting nanospheres comprises the following steps: synthesizing magnetic nanospheres with a particle size of 200 nm by a hydrothermal method; coating a silicon layer with a thickness of 20 nm on the surfaces of the magnetic nanospheres by a gel-sol method; adding the silicon-coated magnetic nanospheres, functional monomers, template molecules, and a cross-linking agent into a Tris-HCl buffer to obtain a reaction solution; performing polymerization of the reaction solution at a certain temperature to obtain polymers; eluting the polymers by an eluate to obtain the temperature sensitive magnetic western-blotting nanospheres. The advantages of the invention are that: the temperature sensitive magnetic western-blotting nanospheres combine the specific recognition of western blotting, the rapid separation performance of magnetic nanospheres in an external magnetic field, and the temperature responsibility of temperature sensitive materials; the preparation method is simple; the conditions are mild; the price is low; and a new feasible method is provided for the selective removal and enrichment of high-abundance protein components in a complex biological system.

The invention relates to a method for detecting a low-abundance protein in blood plasma through surface-enhanced Raman spectroscopy (SERS). The method comprises the following steps: adding a standard blood plasma sample into a centrifugal column of a hydrophobic polymer, performing vibration in a shaking table, and performing centrifugation to obtain a low-abundance protein solution; mixing the low-abundance protein solution with a blotting immobilized matrix and the standard blood plasma sample, and performing incubation to obtain an enrichment blood plasma low-abundance protein blotting immobilized matrix; adding glacial acetic acid and SERS active metal nano-particles; performing incubation to obtain a low-abundance protein SERS enhanced matrix mixed solution; performing SERS detection on the mixed solution; through the operation, acquiring blood plasma low-abundance protein SERS data of a cancer patient with an unknown cancer type; and determining the unknown cancer type by comparing the data with Raman spectral databases of healthy people and known cancer types. The method has the advantages of short detection time, low required power and good economic and social benefits; the immobilized matrix is low in cost; an enrichment process is simple and easy to operate.

The invention belongs to the fields of material science and engineering and biological separation engineering, and particularly relates to a quantum dot-based protein imprinted polymer microsphere as well as preparation and application thereof. The CdTe quantum dot is directly used as a functional monomer through taking mercaptoacetic acid (TGA) and glutathione (GSH) together as a stabilizer, protein is a template molecule, dopamine is a cross-linking agent, and the imprinted polymer microsphere is obtained through the auto polymerization of the dopamine. The quantum dot-based protein imprinted polymer microsphere as well as the preparation and the application thereof provided by the invention have the advantages that fluorescence signals of the quantum dots are utilized for detection, and the functionalized quantum dots are directly taken as functional monomers, so that the quantum dots are contained in imprinted holes. Therefore, the detection sensitivity can be improved. Meanwhile, an imprinted polymer layer is obtained through the auto polymerization of the dopamine, and the imprinting process is further simplified. The quantum dot-based protein imprinted polymer microsphere is used for analysis and detection on phycocyanin in a sea water and lake water sample and bovine hemoglobin in a bovine urine sample, the adding standard recovery rate can reach 90 percent or higher, and the reuse performance is good.

Methods for preparation of molecularly imprinted polymers and their use for detection of proteins and / or polypeptides in a sample are disclosed. The methods of preparation are based on selecting from available data bases an amino acid sequence of a protein / polypeptide target molecule; cleaving the sequence in-silico with at least one cleaving agent, producing fragments with known composition; selecting at least one such fragment comprising a unique epitope; preparing a synthetic peptide representing the unique epitope; and preparing a molecularly imprinted polymer comprising specific binding sites for the synthetic peptide. For detection of the target protein in a sample, the same cleaving agent used for the in-silico cleavage is used to cleave the target protein to form the specific peptide fragments to which the MIP is specific.

The invention discloses a fabrication method for a protein electrochemical imprint sensor of a modified magnetic electrode by a one-step deposition method, and relates to the technical field of electrical analysis chemistry and protein recognition sensors. The fabrication method comprises the steps of 1) preparing a graphene@Fe3O4 nanometer composite material; 2) fabricating a graphene@Fe3O4@Au modified magnetic electrode by the one-step electrodeposition method; 3) introducing boric acid, amino and carboxyl active group into a surface of the modified magnetic electrode so that a plurality ofrecognition sites are provided for protein imprint; 4) pre-assembling protein; 5) performing electrochemical polymerization on a protein imprint membrane; and 6) washing template protein with an eluent so as to build the protein molecular imprint electrochemical sensor. The magnetic modified electrode fabricated by the method is beneficial for introducing the active group, increasing the recognition sites, improving selectivity, expanding surface area and improving conductivity of electrode surface, the built protein imprint electrochemical sensor is high in sensitivity and can be used repeatedly, and target protein can be specially recognized.

The invention belongs to a preparation method and application of a bovine VCAM-1 polyclonal antibody. The method comprises the following steps of (1) RNA (ribonucleic acid) extraction; (2) reverse transcription PCR (polymerase chain reaction); (3) primer design and synthesis; (4) gene fragment amplification; (5) gene clone; (6) prokaryotic expression vector building; (7) expression and identification of DNA (deoxyribonucleic acid) recombinant plasmid in escherichia coli; (8) recombinant protein purification; (9) protein blotting; (10) recombinant protein dialysis and concentration determination; (11) immunizing antigen preparation; (12) immunizing and blood collection; (13) serum separation. The polyclonal antibody provided by the invention can be used for identifying the bovine recombinant VCAM-1 protein and natural recombinant VCAM-1 protein; the valence of the VCAM-1 polyclonal antibody is high; the specificity is high. The antibody particularly aims at the bovine natural VCAM-1 protein.

The invention discloses an experimental method of a long non-coding RNA up-regulation gene in myocardial hypertrophy; the method is characterized in that the levels of lncRNATUG1, miR-497 and myocardial cell enhancement factor 2CmRNA are evaluated through qRT-PCR (quantitative reverse transcription-polymerase chain reaction), western blot measurement is carried out to determine the expression of MEF2C protein, and the endogenous interaction among TUG1, miR-497 and MEF2C is proved through a dual-luciferase reporter gene and RNA immunoprecipitation experiment. The overexpression of the MiR-497 mediates the protective effect of TUG1 knock-down in the cardiac hypertrophy induced by AngII. In addition, the TUG1 regulates the expression of MEF2C by spongizing miR-497. The knock-down of the TUG1 at least partially rescues AngII-induced myocardial hypertrophy by targeting the miR497 / MEF2C axis, and highlights a novel and promising therapeutic target for treating the myocardial hypertrophy.

Nanocrystals having a ZnTe core and methods for making and using them to construct core-shell nanocrystals are described. These core-shell nanocrystals are highly stable and provide quantum yields and stability suitable for applications such as flow cytometry, cellular imaging, and protein blotting, medical imaging, and other applications where cadmium toxicity is an issue.

The invention discloses a non-closed protein blotting method for quickly detecting low-abundance proteins.The non-closed protein blotting method includes steps of (1), separating total proteins by the aid of gel in a gradient manner; (2), blotting the total proteins separated in the gradient manner on PVDF (polyvinylidene fluoride) films from the gel; (3), soaking the PVDF films with the blotted total proteins in 100% methanol; (4), drying the PVDF films treated by the 100% methanol; (5), carrying out color development detection on the target proteins in the dried PVDF films.The total proteins are extracted from samples.The non-closed protein blotting method for quickly detecting the low-abundance proteins has the advantages that the low-abundance protein detection efficiency can be improved, and practical, sensitive and speedy detection means can be provided.

The invention relates to novel application of emodin in the field of pharmacy, i.e. the application of emodin in preparing medicaments for treating P2X3 mediated neuropathic pains / nervous system diseases. In experiments, the emodin is observed to have an effect on inhibiting algesia behavioral reaction and is further observed to have an effect on inhibiting the mRNA and protein expression of nerve cell P2X3 receptors of rat dorsal root ganglia in a neuropathic pain model and rat trigeminal ganglion in a trigeminal neuralgia model by applying the technologies of immunohistochemistry, in-situ hybridization, RT-PCR (Reverse Transcription-Polymerase Chain Reaction), protein blotting and the like. The experiments prove that the mechanism of the effect of the emodin on inhibiting chronic pains (neuropathic pains) and pains transmitted by trigeminal ganglion nerve cells is to block algesia information transmission mediated by a primary sensory nerve cell P2X3 receptor. The invention provides a novel method for preventing and treating the pains mediated by the neuropathic pains and / or the trigeminal ganglion nerve cells; meanwhile, the invention shows that the emodin has the effect on influencing the P2X3 receptor, which is beneficial to medicinal application on preventing and treating nerve system diseases related to the P2X3 receptor.

The invention discloses a method for preparing a protein imprinted polymer by removing template molecules under electric field assisted actions, which has the advantages of time saving, labor saving and high efficiency. The method comprises the following steps: immobilizing a protein polymer onto an electrode as work electrode, wherein the work electrode, a reference electrode and a counter electrode constitute a tri-electrode system; putting the tri-electrode system into a protein polymer template molecule eluting solution, and applying a constant potential for 30-120 minutes between the work electrode and reference electrode to obtain the protein imprinted polymer, wherein if the pH value of the protein polymer template molecule eluting solution is lower than the isoelectric point of the protein, the applied potential is 0.1-1.0V, or else, the applied potential is -1.0 to -0.1V; and drying the obtained protein imprinted polymer in nitrogen.

The present invention relates to a rabbit monoclonal antibody that binds to human Id3 protein and / or mouse Id3 protein with high specificity and high affinity. The antibody has a binding constant, measured with respect to human Id3 protein or mouse Id3, of greater than 1×108 / molar. The antibody has no substantial cross-reactivity to other family Id proteins such as Id1, Id2, or Id4, or other endogenous proteins present in the cells that express Id3 protein. The specificity and high affinity of the rabbit monoclonal antibodies of the present invention allows sensitive and specific detection and / or quantitation of human or mouse Id3 protein in biological samples. The antibodies are useful in immunochemical-based assays such as ELISA, western blot, and immunohistochemical staining.

The invention belongs to the field of separating materials, and particularly relates to a preparation and use method of a protein molecule imprinting separating medium. A template protein molecule is connected to the surface of a matrix such as silica gel, a high molecule and the like through a physical or chemical method; a protein part combined with the surface of the matrix is further embedded by using a partial burying technology; unstable partially embedded proteins are cleared away; the residual proteins float on the surface of the matrix; and the residual proteins and imprinting points on the surface of the matrix achieve a stereo imprinting effect. Such a protein imprinting separating medium can perform high-selectivity separation on a template protein. The invention has the advantages of simple separating material preparation process and high selectivity.

The invention discloses a method for raising expression of carbamoyl phosphate synthetase in HepGL liver cancer cells. The method comprises steps: CPS1 genome DNAs extracted from HepGL liver cancer cells are subjected to bisulfite treatment; the amplified CPS1 promoter area of the treated DNAs is subjected to methylation specific gene amplification; the amplified methylation sequences are subjected to sequencing; TALENs expression plasmids and non-methylation homologous plasmids which are homologous with the methylation loci are constructed according to base sequences around methylation loci, wherein, the homologous non-methylation sequence in the homologous plasmids is a mutation sequence wherein CG is mutated into CA; the TALENs expression plasmids and the homologous plasmids are subjected to transfection into HepGL liver cancer cells, HepGL liver cancer cells with transfection generation are screened, and subjected to Q-PCR and western blotting processing, and the expression amounts of mRNA of CPS1 and proteins in the cells are detected. The method can enhance the ammonia metabolism capability of HepGL liver cancer cells.

The invention provides a thermo-sensitive western blot sensing microsphere based on a quantum dot and a preparation method of same and belongs to the technical field of material science and bio-separation engineering. The method includes the steps of: 1) with a functional protein molecule as a template molecule, combining the functional protein molecule with CdTe quantum dots, which are co-modified with glutathione-thioglycollic acid (GSH-TGA), through electrostatic charge effect by means of amino groups and carboxyl groups, which are rich in the functional protein molecule; 2) in a non-oxygenenvironment, initiating polymerization by means of potassium persulfate (KPS), wherein thermo-sensitive isopropyl acrylamide (NIPAM) is used as a functional monomer and N,N-methylene bisacrylamide (MBA) is used as a crosslinker, so that through the free radical polymerization, the thermo-sensitive western blot sensing microsphere based on the quantum dot is produced. Through mechanism of electrontransfer between the functional protein and the quantum dot, fluorescent sensing of the microspheres to the template protein is achieved, and the synthesized western blot microspheres has performances such as thermo-sensitive response, fluorescent detection, etc.

The invention relates to a preparation method and an adsorption device of a protein imprinted polymer adsorbent. The preparation method comprises the following steps: step 1: mixing and dispersing siloxane monomers uniformly under conditions lower than normal temperature and nitrogen atmosphere; step 2 : amination and aldehydelation of polymer microspheres, and then loading porogen; step 3: uniformly dispersing the protein aqueous solution and the mixture obtained in step 2 in the mixture obtained in step 1, adding acid, and carrying out condensation reaction; step 4: The mixture obtained in Step 3 was filtered, washed until neutral and dried to prepare a polymer adsorbent for western blotting. The preparation method is simple to operate, and the obtained western blotting polymer adsorbent has a high adsorption rate of toxin, and is especially suitable for uremic hemoperfusion.

The invention relates to the new usage of puerarin in the field of pharmaceutical products, in other words, it relates to the application of puerarin in the preparation of drugs for P2X3 mediated pain / nervous system diseases. The experiment shows that puerarin can inhibit pain-related behavioral responses, subsequently immunohistochemical analysis, in situ hybridization, RT-PCR, protein blotting and other techniques were utilized to observe the inhibition of puerarin on mRNA and protein expression of P2X3 receptor in dorsal root ganglion of rats with neuropathic pain and in dorsal root ganglion and sensory nerve ending in the burn model of rats, it was found by using whole-cell patch clamp technique that puerarin can significantly reduce the electric current activated by P2X3 receptor agonist in the neuron of dorsal root ganglion of rats with neuropathic pain. The experiment proves that the mechanism for the inhibitory effects of puerarin on acute and chronic pain is to block the transmission of pain sense information that is mediated by P2X3 receptor in primary sensory neurons. The invention provides a kind of new method for the prevention and treatment on acute and chronic pain, and it also indicates that puerarin can act as a antagonist for P2X3 receptor, which will be helpful for the application of drugs in the prevention and treatment on P2X3 receptor involved nervous system diseases.

The invention relates to a protein macromolecule imprinted calcium phosphate / calcium alginate hybrid microsphere and a preparation method thereof. The composition and weight percentage of calcium phosphate / calcium alginate hybrid microspheres for western blotting are as follows: sodium alginate 20%-40%, diammonium hydrogen phosphate 1%-10%, calcium chloride 50%-65%, Protein 0.5-10%. The calcium phosphate / calcium alginate hybrid microspheres prepared by the present invention show that the adsorption capacity of the imprinted microspheres is 1.8 to 5 times that of the non-imprinted microspheres, that is, the microspheres prepared without adding protein, according to the protein adsorption test results. Conductivity titration and infrared spectroscopy tests show that diammonium hydrogen phosphate and sodium alginate can interact with CaCl2 to produce new hybrid components. The invention has the advantages of simple preparation method, easy control of the reaction process, etc., solves the shortcomings of pure calcium alginate such as low mechanical strength and imprinting efficiency, unstable performance, difficult diffusion of protein macromolecules, and the like, and has broad application prospects.

The invention relates to a method for simultaneously rotating two films and marking two proteins, namely TRX and procaspase12. The method includes the steps of total protein extraction of cells, protein quantifying, gel electrophoresis, protein blotting, simultaneously marking of antibodies, X-ray film developing, and the like. The invention aims at building up a method which is characterized in that two PVDF films are used for simultaneously marking and detecting the two target proteins by using different antibodies after SDS-PAGE and protein blotting are carried out on the total protein in cells, meanwhile, the method is compared with the traditional method which has the steps of stripping films and then marking antibodies, and the accuracy, the reproducibility, the stability, and the like of the method are evaluated. The invention is simple and easy to carry out, can well represent the test result, and provides a method which is simpler and easier and saves time and samples, thereby having actual application value.

The invention relates to a method for detecting a low-abundance protein in blood plasma through surface-enhanced Raman spectroscopy (SERS). The method comprises the following steps: adding a standard blood plasma sample into a centrifugal column of a hydrophobic polymer, performing vibration in a shaking table, and performing centrifugation to obtain a low-abundance protein solution; mixing the low-abundance protein solution with a blotting immobilized matrix and the standard blood plasma sample, and performing incubation to obtain an enrichment blood plasma low-abundance protein blotting immobilized matrix; adding glacial acetic acid and SERS active metal nano-particles; performing incubation to obtain a low-abundance protein SERS enhanced matrix mixed solution; performing SERS detection on the mixed solution; through the operation, acquiring blood plasma low-abundance protein SERS data of a cancer patient with an unknown cancer type; and determining the unknown cancer type by comparing the data with Raman spectral databases of healthy people and known cancer types. The method has the advantages of short detection time, low required power and good economic and social benefits; the immobilized matrix is low in cost; an enrichment process is simple and easy to operate.

The invention relates to a new purpose of resveratrol in the pharmaceutical field, that is, an application of resveratrol in the preparation of medicines for treating P2X3-mediated chronic pain diseases. Experimental observation results show that resveratrol can inhibit pain related behaviors, and observation results of experiments applied with technologies of immunohistochemistry, in situ hybridization, RT-PCR, protein blotting and the like show that resveratrol can inhibit the mRNA expression and the protein expression of a P2X3 acceptor of the dorsal root ganglia from a neuropathic pain model rat. Experiments confirm that the mechanism of the inhibition effect of resveratrol to chronic pains is the antagonism of the primary sensory ganglion P2X3 acceptor-mediated pain information transmission. According to the invention, a new chronic pain (neuropathic pain) control method is ascertained, and simultaneously a case that resveratrol has effects of P2X3 acceptor antagonists is disclosed, so above observations of the invention are in favor of the application of resveratrol in the preparation of purine X3 acceptor (P2X3 acceptor) specificity antagonists.

The present invention relates to a fluorescent cell binding assay combining pre-labeling and Western blotting. Intact cells are incubated with pre-labelled binders preferably followed by SDS PAGE (sodium dodecylsulphate polyacrylamide gel) separation and Western blotting. More closely, the invention relates to a cell binding assay in which the degree or amount of binding of one or more cell interacting protein or protein component to the cell surface is measured with the ability to correlate the degree of cell binding to the sample load / total number of cells.

The invention discloses the novel use of a VEGF antibody and a VEGF receptor antagonist in the fields of pain-killing vaccines and pharmacy, namely the use of the VEGF antibody or the VEGF receptor antagonist in the preparation of an acute / chronic pain killing vaccine and a medicament for treating P2X3 and P2X2 / 3 purine receptor-mediated nerve system diseases. Through experiments it is observed that the VEGF antibody can inhibit the algetic behavior response; and by using immunohistochemistry, in-situ hybridization, RT-PCR, protein blotting and other techniques, it is observed that the VEGF antibody can inhibit the expression of mRNAs and proteins of the P2X2 and P2X3 receptors, VEGF and F1k-1 / VEGFR2 of rat dorsal root ganglia of a model of neuropathic pain. Experiments prove that the pain killing mechanism of the VEGF antibody is that the pain is killed by neutralizing the VEGF or antagonist VEGF receptor to reduce pathologic pains and by blocking the pain information transmission of dorsal root neurons. The invention provides a new method for preventing and treating acute and chronic pains. Meanwhile, it is shown that the VEGF antibody can inhibit the expression of the P2X2 and P2X3 receptors, which is favorable for the use of the VEGF antibody and the VEGF receptor antagonist in the prevention and treatment of nerve system diseases related to the P2X2 and P2X3 receptors.

The invention discloses a fabrication method for a protein electrochemical imprint sensor of a modified magnetic electrode by a one-step deposition method, and relates to the technical field of electrical analysis chemistry and protein recognition sensors. The fabrication method comprises the steps of 1) preparing a graphene@Fe3O4 nanometer composite material; 2) fabricating a graphene@Fe3O4@Au modified magnetic electrode by the one-step electrodeposition method; 3) introducing boric acid, amino and carboxyl active group into a surface of the modified magnetic electrode so that a plurality ofrecognition sites are provided for protein imprint; 4) pre-assembling protein; 5) performing electrochemical polymerization on a protein imprint membrane; and 6) washing template protein with an eluent so as to build the protein molecular imprint electrochemical sensor. The magnetic modified electrode fabricated by the method is beneficial for introducing the active group, increasing the recognition sites, improving selectivity, expanding surface area and improving conductivity of electrode surface, the built protein imprint electrochemical sensor is high in sensitivity and can be used repeatedly, and target protein can be specially recognized.

The present invention relates to a method for identification of specific target proteins in a protein sample following a detection procedure, such as a Western blotting procedure, wherein the membrane is probed with at least two primary antibodies directed against the same and / or different epitopes of the same target protein, and wherein specific binding to the target protein in a sample is differentiated from unspecific binding to the target protein by comparing the resulting sample patterns, such as bands or spot patterns, with each other.In a further step signals from the true target proteins are enhanced while signals resulting from unspecific binding are diminished.

The invention discloses a preparation method of a molecularly imprinted composite material for enriching and separating glycoproteins, belonging to the fields of material science and engineering and biological separation engineering. In this method, the surface of graphene oxide is firstly functionalized with boric acid, the template glycoprotein is fixed on the surface of graphene oxide through boron affinity, and the surface imprinting technology is used to form a protein imprinted layer on the surface of graphene oxide, and the template is eluted to obtain molecular imprinting. composite material. The molecularly imprinted composite material prepared by this method has a very good ability to specifically recognize the template glycoprotein, and also has the advantages of high binding capacity, strong anti-interference ability, fast binding and elution speed, and can be used for complex practical applications. The low-concentration glycoproteins in the sample are enriched, separated and detected.