33 results about "Two-dimensional gel electrophoresis" patented technology

The present invention provides methods for determining the methylation status of CpG-containing dinucleotides on a genome-wide scale using infrequent cleaving, methylation sensitive restriction endonucleases and two-dimensional gel electrophoretic display of the resulting DNA fragments. Such methods can be used to diagnose cancer, classify tumors and provide prognoses for cancer patients. The present invention also provides isolated polynucleotides and oligonucleotides comprising CpG dinucleotides that are differentially methylated in malignant cells as compared to normal, non-malignant cells. Such polynucleotides and oligonucleotides are useful for diagnosis of cancer. The present invention also provides methods for identifying new DNA clones within a library that contain specific CpG dinucleotides that are differentially methylated in cancer cells as compared to normal cells.

The present invention relates to protein markers for diagnosing stomach cancer and a diagnostic kit using the same, more precisely protein markers screened by two-dimensional gel electrophoresis and bioinformatics and a diagnostic kit using the same. The markers of the invention can be effectively used for diagnosing stomach cancer and evaluating the extent of progress of the cancer by confirming the expression levels of those marker proteins whose expressions differ in stomach cancer patients from in normal healthy people.

Disclosed are methods and apparatus for separation of biomolecules via two-dimensional gel electrophoresis, methods and apparatus for immunoblotting separated biomolecules, and methods for the use of biomolecules processed via the methods and apparatus of the present invention, including use in a clinical setting. The methods and apparatus for separation of biomolecules via two-dimensional gel comprises vertical agarose gel electrophoresis in the first dimension, and the electrophoresis of a novel non-denaturing 3-35% concave gradient polyacrylamide gel in the second dimension. This novel gel can be cast in a modified gel caster that can facilitate the pouring of multiple gels simultaneously. The methods and apparatus for immunblotting are useful with any type of immunoblotting, including Western blot, Northern blot, and Southern blot analyses. These methods and apparatus provide safe, efficient and cost-effective immunoblots, while facilitating the reduction of exposure to toxic or radioactive materials, as well as the disposal of those materials.

The invention provides a buildup method of a protein difference expression atlas when the committed differentiation of stem cells is induced by drugs, which detailedly establishes the protein difference expression atlas formed by that the stem cell is induced by the ICA and directionally differentiated to be a cardiac muscle cell; a model that the drug induces the stem cell to be directionally differentiated to be the cardiac muscle cell is established so as to prepare a two-dimensional electrophoresis protein sample; electrophoresis is gelled bidirectionally, images are collected and analyzed so as to confirm the difference protein. A comparison protein atlas is used for screening and identifying the difference expression protein when the ES cell is directionally differentiated to be the cardiac muscle cell by the inducing of the ICA; the difference expression protein is used as a drug effect target and applied to the preparation of a novel inducer which has high-efficiency and low-toxicity and is used for prompting the stem cell to be differentiated.

The invention relates to an extraction method of protein from animal tissues, in particular to an extraction method of total protein from a rumen epithelial tissue of a milk cow. The method comprises the steps of putting a rumen epithelial tissue sample of the milk cow in a precooled mortar, adding liquid nitrogen, grinding into powder, adding an acetone solution of precooled trichloroacetic acid, oscillating, eddying, uniformly mixing for overnight aging, centrifugally collecting sediment, freeze-drying the tissue sediment, adding lysate, incubating and eddying at intervals, and centrifugally collecting supernate, namely the total protein of the rumen epithelial tissue of the milk cow. For the total protein of the rumen epithelial tissue of the milk cow, extracted by the method, a total protein mixture can be separated by a two-dimensional gel electrophoresis technology according to an isoelectric point and molecular weight of the protein, the relative abundance of the protein is acquired, the isoelectric point, the molecular weight and a relative transcript level of each protein can be clearly and visually shown in a gel atlas, and the method has the advantages of more obvious integrity and visualization in comparison with the prior arts such as a high efficiency liquid chromatography separation method, an enzyme-linked immunosorbent assay and immunoblotting.

Disclosed are methods and apparatus for separation of biomolecules via two-dimensional gel electrophoresis, methods and apparatus for immunoblotting separated biomolecules, and methods for the use of biomolecules processed via the methods and apparatus of the present invention, including use in a clinical setting. The methods and apparatus for separation of biomolecules via two-dimensional gel comprises vertical agarose gel electrophoresis in the first dimension, and the electrophoresis of a novel non-denaturing 3-35% concave gradient polyacrylamide gel in the second dimension. This novel gel can be cast in a modified gel caster that can facilitate the pouring of multiple gels simultaneously. The methods and apparatus for immunblotting are useful with any type of immunoblotting, including Western blot, Northern blot, and Southern blot analyses. These methods and apparatus provide safe, efficient and cost-effective immunoblots, while facilitating the reduction of exposure to toxic or radioactive materials, as well as the disposal of those materials.

A new instrumental design is provided for in-gel detection and quantification of proteins. This new platform, called Cumulative Time-resolved Emission 2-Dimensional Gel Electrophoresis, utilizes differences in fluorescent lifetime imaging to differentiate between fluorescence from specific protein labels and non-specific background fluorescence, resulting in a drastic improvement in both sensitivity and dynamic range compared to existing technology. The platform is primarily for image acquisition of two-dimensional gel electrophoresis, but is also applicable to protein detection in one-dimensional gel systems as well as proteins electroblotted to e.g. PVDF membranes. Given the increase in sensitivity of detection and dynamic range of up to 5-6 orders of magnitude compared to existing approaches, the described invention represents a technological leap in the detection of low abundance cellular proteins, which is desperately needed in the field of biomarker discovery.

The invention discloses a preparation method of a micro-fluidic chip interface for two-dimensional protein electrophoretic separation, and relates to a micro-fluidic chip. The method comprises the following steps of: preparing polyacrylamide gel in a vertical channel of the micro-fluidic chip and filling a buffering aqueous solution; introducing an organic phase through a horizontal channel of the micro-fluidic chip and filling the horizontal channel; introducing an organic phase into which tetraisopropyl titanate is dissolved and filling the horizontal channel; draining an organic solution of tetraisopropyl titanate out of the horizontal channel, and cleaning the horizontal channel; adding an isoelectric focusing buffer solution into which proteins and amphoteric electrolytes are dissolved into the horizontal channel, applying electric fields to the two ends of the horizontal channel for performing isoelectric focusing, and realizing first-dimension separation of proteins in the horizontal channel according to respective different isoelectric points; and after finishing isoelectric focusing, applying electric fields to the two ends of the horizontal channel for performing second-dimension gel electrophoresis, making micropores on a TiO2 film pass through an electric field, and making the proteins enter a second-dimension channel by passing through the micropores on the TiO2 film under the action of the electric field for performing two-dimension separation.

A new instrumental design is provided for in-gel detection and quantification of proteins. This new platform, called Cumulative Time-resolved Emission 2-Dimensional Gel Electrophoresis, utilizes differences in fluorescent lifetime imaging to differentiate between fluorescence from specific protein labels and non-specific background fluorescence, resulting in a drastic improvement in both sensitivity and dynamic range compared to existing technology. The platform is primarily for image acquisition of two-dimensional gel electrophoresis, but is also applicable to protein detection in one-dimensional gel systems as well as proteins electroblotted to e.g. PVDF membranes. Given the increase in sensitivity of detection and dynamic range of up to 5-6 orders of magnitude compared to existing approaches, the described invention represents a technological leap in the detection of low abundance cellular proteins, which is desperately needed in the field of biomarker discovery.

The invention discloses an identification method of intracellular and extracellular proteomes of penicillium during fermentation. The identification method comprises the following steps of: (1) extracting intracytoplasmic proteins; (2) extracting extracytoplasmic proteins; (3) carrying out two-dimensional gel electrophoresis; (4) analyzing a two-dimensional gel image; and (5) identifying the proteins by using MALDI (Matrix-Assisted Laser Desorption Ionization)-TOF (Time-Of-Flight)-MS (Mass Spectrometry). The protein level closely related to penicillin synthesis in a penicillin fermentation process, and a relation between the protein level and the penicillin production level can be found quickly and accurately in high flux by using the identification method, differentially expressed proteins are found out and provide a target spot for further modifying a penicillium metabolic path, thus a certain theory is provided for obtaining high production strains and improving the yield of penicillin, and a new method and a concept are provided for modifying other industrial antibiotic strains.

The invention relates to an application of human derived HSF2 as the specific diagnosis molecular marker of ulcerative colitis, and belongs to the technical field of biomedicine. The technical scheme comprises three parts: (1) detecting differential proteins in serums of UC patients and healthy volunteers through a proteomics two-dimensional gel electrophoresis technology and a mass spectrum technology; (2) detecting the HSF2 expression level in colonic mucosa of UC patients in different degrees through an immunohistochemical method; (3) detecting the HSF2 expression level in colonic mucosa of UC patients in different degrees through a real-time quantitative PCR technology and a western blotting technology. 40 UC patient serums and 40 healthy volunteer serums have been compared by the method mentioned above, and the results show that the expression of HSF2 in the UC patient serums prominently increases. Moreover, an immunohistochemical method, a real-time quantitative PCR technology and a western blotting technology are adopted to detect the HSF2 expression in the colonic mucosa of UC patients, and the results show that the HSF2 expression increases as the UC disease goes worse. Thus, the HSF2 can be used as a molecular marker to specifically diagnosing ulcerative colitis.

The invention discloses a protein complex or compound three-dimensional gel electrophoresis separating method. The method comprises the following steps: 1) separating the protein complex by non-denatured polyacrylamide gel electrophoresis method and then dyeing the protein complex; 2) chopping the gel strips containing the target protein complex or compound obtained in step 1), and decoloring and dehydrating; and 3) soaking the gel strips processed in step 2) by protein denaturation solution, adequately pulverizing and directly performing separation by two-dimensional gel electrophoresis method. The method in the invention dispenses with protein electroelution and protein deposition operating procedures in traditional protein complex or compound three-dimensional gel electrophoresis method, thus avoiding protein loss and protein in vitro chemical decoration; and the method in the invention is simple to operate and needs no additional equipment. Proved by experiments, the method in the invention achieves good protein complex or compound electrophoresis separating effect.

The invention provides a method for identifying a human growth hormone protein existence form biomarker spectrum, which comprises the following steps: collecting human growth hormone secreting pituitary adenoma and normal pituitary tissue samples, respectively extracting tissue proteins, carrying out two-way gel electrophoresis, western blot and coomassie brilliant blue staining, scanning a visual PVDF membrane and a two-way gel into a digital image, digesting corresponding two-way gel protein spots with trypsin, purifying, and identifying a GHP biomarker spectrum through mass spectrum identification and bioinformatics analysis; and identifying post-translational modifications and shear variations on GHP by using quantitative phosphorylated proteomics, ubiquitinated proteomics, and acetylated proteomics analysis in combination with bioinformatics. According to the invention, the GHP change mode between the growth hormone secreting pituitary adenoma and normal pituitary tissues can be identified, 46 kinds of GHPs are identified in the growth hormone secreting pituitary adenoma, 35 kinds of GHPs are identified in the normal pituitary tissues, and 11 kinds of GHPs only appear in the growth hormone secreting pituitary adenoma tissues.

The invention relates to a method for utilizing a proteomic technology to screen a drug action target position of cyclosporine A in an immunized T cell. According to the method for screening the drug action target position of cyclosporine A in the immunized T cell, the proteomic technology is used for screening the drug action target position of cyclosporine A in the immunized T cell and the method comprises following steps: step 1) establishing sample groups of the action of cyclosporine A immunizing the T cell; step 2) preparing two-dimensional electrophoresis protein samples of the immunized T cell; step 3) carrying out two-dimensional gel electrophoresis; step 4) collecting images and analyzing to obtain the result. The method for screening the drug action target position of cyclosporine A in the immunized T cell, disclosed by the invention, is clear and definite in operation through, and true and reliable in result, overcomes the defects that with adoption of the conventional screening method, the screening result is high in error and low in accuracy as the analysis data are huge and complicated, and provides the detection result with pharmacologic significance and guiding function for clinical medication.

The invention discloses a prostate cancer metabonomics marker and a screening method thereof, blood is subjected to abundance protein removal treatment and then purified by a settling agent, then fluorescent staining and two-way gel electrophoresis are performed to obtain the prostate cancer metabonomics marker, and carboxyl on carboxymethyl chitosan and amino on the surface of silicon dioxide are subjected to dehydration condensation to obtain the prostate cancer metabonomics marker. The preparation method comprises the following steps: preparing modified chitosan, carrying out hydrolytic polymerization on aluminum chloride and ferric chloride, carrying out loading by using the modified chitosan as a carrier to prepare composite chitosan, blending the composite chitosan with starch, and grafting with an acrylamide copolymer to prepare the settling agent. Compared with a traditional settling agent, the settling agent can more quickly adsorb impurities in plasma to form insoluble precipitates, so that the plasma protein purification efficiency is improved, and the influence of the impurities on the screening accuracy of the metabonomics markers of the adenocarcinoma is avoided.

The invention discloses a method for identifying a glycoprotein sugar chain structure by combining two-dimensional gel electrophoresis and mass spectrometry. The method comprises the following steps:S1, performing specific enrichment on glycoprotein in a biological sample, performing separation through two-dimensional gel electrophoresis, and selecting target glycoprotein isomer gel spots according to the molecular weight and isoelectric point of target glycoprotein; S2, confirming the selected target glycoprotein isomer gel spots by using a two-dimensional gel electrophoresis western blot and a mass spectrum; and S3, performing intra-gel enzymolysis and mass spectrometry on the target glycoprotein isomer gel spots confirmed in the step S2, and identifying the peptide fragment sequence and the sugar chain structure. The method is suitable for separation and structural identification of isomers and modifiers of various protein post-translational modified (glycosylated, ubiquitinated, acetylated and the like) biomarkers, and can significantly improve the sensitivity and specificity of the post-translational modified protein as a diagnostic marker.

The invention provides a biomarker method for identifying the existence form of human pituitary tissue lactation hormone protein. The method comprises the following steps of collecting pituitary adenoma and normal pituitary tissue samples, respectively extracting the tissue proteins, scanning a visual PVDF membrane and bidirectional gel into a digital image through bidirectional gel electrophoresis and western blot silver staining, digesting and purifying the corresponding two-way gel protein spots, and identifying the difference of the existing form proportions of different pituitary tissue lactation hormone proteins of different pituitary tumor sub-types through mass spectrum identification and bioinformatics analysis. According to the present invention, the existence forms of the human pituitary tissue lactation hormone protein and the differential expression profiles of the prolactin proteins in different pituitary tumors are identified, so that the development of drugs for blocking prolactin signal channels for clinical treatment is facilitated.

The invention relates to a method for utilizing a proteomic technology to screen a drug action target position of mizoribine in an immunized T cell. According to the method for screening the drug action target position of mizoribine in the immunized T cell, the proteomic technology is used for screening the drug action target position of mizoribine in the immunized T cell and the method comprises the following steps: step 1) establishing sample groups of the action of mizoribine immunizing the T cell; step 2) preparing two-dimensional electrophoresis protein samples of the immunized T cell; step 3) carrying out two-dimensional gel electrophoresis; step 4) collecting images and analyzing to obtain the result. The method for screening the drug action target position of mizoribine in the immunized T cell, disclosed by the invention, is clear and definite in operation through, and true and reliable in result, overcomes the defects that with adoption of the conventional screening method, the screening result is high in error and low in accuracy as the analysis data are huge and complicated, and provides the detection result with pharmacologic significance and guiding function for clinical medication.

The invention relates to a method for utilizing a proteomic technology to screen a drug action target position of mizoribine in an immunized T cell. According to the method for screening the drug action target position of mizoribine in the immunized T cell, the proteomic technology is used for screening the drug action target position of mizoribine in the immunized T cell and the method comprises the following steps: step 1) establishing sample groups of the action of mizoribine immunizing the T cell; step 2) preparing two-dimensional electrophoresis protein samples of the immunized T cell; step 3) carrying out two-dimensional gel electrophoresis; step 4) collecting images and analyzing to obtain the result. The method for screening the drug action target position of mizoribine in the immunized T cell, disclosed by the invention, is clear and definite in operation through, and true and reliable in result, overcomes the defects that with adoption of the conventional screening method, the screening result is high in error and low in accuracy as the analysis data are huge and complicated, and provides the detection result with pharmacologic significance and guiding function for clinical medication.

Plasma samples of ovarian and breast cancer patients were used to search for markers of cancer, using two-dimensional gel electrophoresis and MALDI TOF mass spectrometry. Truncated forms of cytosolic serine hydroxymethyl transferase (cSHMT), T-box transcription factor 3 (Tbx3) and utrophin were aberrantly expressed in samples from cancer patients, as compared to samples from noncancer cases. Aberrant expression of proteins was validated by immunoblotting of plasma samples with specific antibodies to cSHMT, Tbx3 and utrophin. A cohort of 79 breast and 39 ovarian cancer patients, and 31 individuals who were either healthy or had noncancerous conditions was studied. We observed increased expression of truncated cSHMT, Tbx3 and utrophin in plasma samples obtained from patients at early stages of disease. The results indicate that cSHMT, Tbx3, utrophin and truncated forms thereof can be used as components of multiparameter monitoring of ovarian and breast cancer.

A biochemical marker for the diagnosis of a central nervous system leukodystrophic genetic disorder, e.g., Childhood Onset Ataxia and Central Nervous System Hypomyelination (CACH) / Vanishing White Matter Disease (VWM) has been discovered herein. Such a marker has been found in the cerebrospinal fluid (CSF) of such patients. A two dimensional gel electrophoresis / mass spectrometry or image analysis of stained transferrin isoforms approach revealed that patients with CACH / VWM have a pronounced deficiency of the basic asialo form of the transferrin compared to the amounts of asialotransferrin normally present in CSF from healthy controls or other CNS disorders. The acidic sialotransferrin isoform is not reduced in these disorders. The transferrin isoform abnormality described in the CSF of patients with CACH / VWM is unique and may be used as a clinical diagnostic biomarker. The rapid (48 hr) and efficient diagnosis of this disorder described herein will have great clinical utility.

The invention provides a method for screening and confirming a kidney-yang deficiency animal model biomarker. The method comprises the following steps: extracting total protein of cancellous bone of a sample; determining the protein concentration; performing two-dimensional gel electrophoresis analysis; detecting gel protein spot and displaying and analyzing differential protein spot; performing MALDI-TOF-MS mass spectrometry for differential protein, retrieving and identifying a protein database; confirming the differential protein; identifying a part of differential protein through Western-Blot and RT-PCR and mutually verifying the detection result for differential protein ELISA in serum; using the differential protein in consistent expression tendency and beyond the normal value scope as a biomarker for identifying a kidney-yang deficiency animal model. The invention is beneficial to the supply of an identifying index to the kidney-yang deficiency in an animal experiment and provides a basis for further discovering the kidney-yang deficiency biomarker.

The invention belongs to biological gene engineering field, especially relates to an Aegilops tauschii high molecular weight glutelin 1Dy12.1#+[t] gene and its uses in biological improvement of wheat quality based on the gene. Glutelin gene is mainly separated using library establishment method and PCR method. In the invention, a new high molecular weight glutelin HMW-GS12.1#+[t] and its gene are separated from Aegilops tauschii using allele specific PCR, single-direction or two dimensional gel electrophoresis and capillary electrophoresis. The gene sequence has high homologue with quality gene 1Dy10, can work in wheat quality improvement.

The invention relates to a method for screening cardiac glycoside drug action targets by proteomics technology, which belongs to the field of medical technology. Including: cardiomyocyte culture and the establishment of cardiac glycoside drugs on cardiomyocyte models, preparation of cardiomyocyte two-dimensional electrophoresis protein samples, two-dimensional gel electrophoresis, image acquisition and analysis, obtaining differentially expressed proteins of cardiac glycoside drugs on cardiomyocytes, After in-gel digestion of the differential proteins, use mass spectrometry to determine the mass fingerprint of the differential protein peptides; enter the sequence database to identify and analyze the differential proteins, and deduce their DNA sequences. Perform mass spectrometry analysis on characteristic proteins, and screen out differential proteins by comparing with mass spectrum libraries; verify the biological functions of differentially expressed proteins. Using proteomics technology to screen the drug targets of cardiac glycosides against heart failure can be directly used to guide the development of new high-efficiency and low-toxicity anti-heart failure drugs.

The invention discloses a method for predicting wheat heterosis by a mitochondrial proteome technology. The invention mainly relates to cultivation of wheat Jing 411, wheat translocation line Ta-6DL / 6VS and its hybridization progeny F1 (Jing 411(female)*Ta-6DL / 6VS(male)). By separating the mitochondrial proteome of the material, and combining a MALDITOFMS technology by utilizing bidirectional gel electrophoresis, obvious difference showing in leaf mitochondrial proteome between wheat hybrid and amphiphilic is discovered, and the difference proteins has four kinds of expression modes: an additional type (ADT), a prejudiced amphiphilic type (PDT), a higher than amphiphilic type (HPDT) and a lower than amphiphilic type (LPDT), wherein expression frequencies of the four modes are respectively 15.2%, 54.5%, 24.2% and 6.1%. Wherein, spot 22 (alcohol dehydrogenase) continuously shows higher than amphiphilic type expression in different growth periods of the wheat hybrid F1 and amphiphilic. The protein can be used for advantage prediction of the hybrid F1.

The present invention discloses a gel electrophoresis chip, comprising a first substrate, a first plurality of parallel gel strips formed on the first substrate, respectively extending along a first direction and having a certain width; and a second plurality of isolation segments formed on the first substrate, respectively located between adjacent gel strips and extending along a second direction different from the first direction, the isolation segments being arranged to form a microwell array together with the gel strips. After the gel electrophoresis chip achieves conventional protein two-dimensional gel electrophoretic separation, protein samples suitable for mass spectrometry analysis are prepared in high throughput, thus greatly reducing the pretreatment time of mass spectrometry analysis, thereby being suitable for proteomic analysis of biological samples.

The invention belongs to the field of protein gel electrophoresis separation, and particularly relates to a charge-mass double-focusing two-dimensional gel electrophoresis separation method and a kit thereof. According to the electrophoresis separation method, under the action of selective coordination of Cu<2+> and amino acid residues in protein under an acidic condition, not only is the protein positively charged due to protonation of the basic amino acid residues, but also similar histone isomers of an amino acid sequence are positively charged differently due to a selective coordination function of metal ions, after direct current is applied, the histone isomers with different positive charges have different migration rates, so that separation is achieved; and after one-dimensional charge separation, SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) can be further performed, and the charge-mass double-focusing two-dimensional gel electrophoresis is used for separation of the histone isomers. The electrophoresis separation method is simple in operation process, high in resolution and low in cost, and defects of high probability of protein precipitation and analysis limitation of the histone isomers during isoelectric focusing electrophoresis in the prior art are overcome.