37 results about "2 Dimensional Gel Electrophoresis" patented technology

An automated, two-dimensional gel electrophoresis technique includes methods and apparatuses for performing a functionally equivalent, automated two-dimensional gel electrophoresis process in an integrated, robotic apparatus.

Removal of abundant proteins from a sample enhances detection and resolution of less abundant proteins in the sample such as in two-dimensional gel electrophoresis. The removal is accomplished by immunosubtraction of several high abundance, interfering or contaminating proteins simultaneously.

The present invention relates to an electrophoresis apparatus comprising a gel chamber for receiving electrophoresis medium, a removable gel system, which is arranged in the gel chamber, having a separation gel for the electrophoretic separation of biological molecules such as nucleic acids or proteins, electric contact elements for generating an electric field through the separation gel, optionally a lid for fastening on the gel chamber, characterized in that the separation gel is delimited at least at one side by a spacer element which is in the form of a collector and comprises a plurality of sample collection containers, which are arranged one next to the other, for fractionating and for collecting the electrophoretically separated molecules. The invention further relates to a method for the electrophoretic separation and collection of biological molecules by way of a two-dimensional gel electrophoresis.

The invention relates to the analysis of complex protein mixtures such as entire proteomes, and in particular the rapid detection of previously unknown or unusually expressed proteins by common enzymatic digestion, subsequent chromatographic separation and analysis of the digestion peptides by mass spectrometry. The invention consists in subjecting fractions of the digestion peptides separated by liquid chromatography to analysis by mass spectrometry, at a time other than that of the chromatography, in a tandem time-of-flight mass spectrometer with ionization by matrix assisted laser desorption (MALDI). This method finds many times more proteins than are found through the procedures predominantly used until now of two-dimensional gel electrophoresis with subsequent time-of-flight mass spectrometry. It also removes the time pressure that dominates the real-time analysis of coupled LC-MS processes, and it allows measurements to be reduced to the interesting proteins by intermediate analysis.

Two-dimensional gel electrophoresis apparatus includes an electrophoresis zone having four edges defined by a plurality of electrodes. A pair of opposed edges are defined by groups of discrete electrodes. Discrete electrodes within each group are electrically isolated from each other while the other pair of opposed edges are used to generate an electrical field. As a result, the electrical field is less distorted than would be the case if each edge was defined by a single elongate electrode. The apparatus can be provided as a cassette with electrodes configured to guide gas generated during electrolysis out of the cassette through apertures, to reduce the build up of combustible gases.

Disclosed is a personalized diagnostic and treatment solution for cardiovascular disease. The invention comprises an extended CVD risk assessment panel, combining tests for traditional and new important risk markers, and methods for devising a personalized treatment plan for a patient via the use of a CVD diagnosis and treatment protocol algorithm. The new important risk markers include HDL subpopulation profile by two-dimensional gel-electrophoresis, plasma sterols, direct measurement of sdLDL-C, determination of CRP molecular forms, glycated albumin as a percentile of total albumin, and other specialized testing pertaining to apolipoprotein E and Factor V Leiden genotyping, NT-proBNP and adiponectin. This solution provides a more complete risk assessment of an individual than merely measuring traditional CVD risk markers, and enables the healthcare practitioner to optimize therapy for patients with or without established CVD. This solution presents the advantages of greater accuracy, savings in time and cost over existing testing and treatment methods.

The present invention relates to the segmentation of two-dimensional gel electrophoresis images (2D images). The method according to the invention associates an initial protein seed candidate with an interface circumscribing said seed and thereafter brings said interface to evolve in accordance with a defined speed function F(x, y). The evolution of the interface is halted by a stopping criterion, C. According to the invention, the speed function can depend on a wide variety of parameters such as the pixel intensity, the curvature of the pixel intensity, the distance to the initial seed, the curvature and / or shape and / or normal direction and / or position of the evolving interface. The stopping criterion depends e.g. on the speed function F and / or the time of arrival T(x, y) and / or the departure time Td for said interface. The invention provides criteria for a specific treatment of saturated spots and to mike sure that interfaces never overlap.

A method for selecting action target of cardiac glucocide medicine by utilizing protein multistudy technique setting up model of cadiocytes culture and action on cadiocytes by cardiac glucocide, preparing protein sample, carrying two ¿C way gel electrophoresis and collecting image for analysis to obtain difference expression protein, using mass spectrum to determine difference protein peptide mass finger atlas to obtain DNA series for selecting out difference protein, verifying biological property of difference expression protein and using protein multistudy technique to select target of cardiac glucocide medicine.

Removal of abundant proteins from a sample enhances detection and resolution of less abundant proteins in the sample such as in two-dimensional gel electrophoresis. The removal is accomplished by immunosubtraction of several high abundance, interfering or contaminating proteins simultaneously.

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.

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.

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.

Methods are provided for separating linear nucleic acid fragments based on their conformation. The methods are based on novel two-dimensional gel electrophoresis techniques. In one aspect the method comprises electrophoresing a sample of nucleic acids in a first dimension said sample through a gel matrix under a first set of pre-determined electrophoresis conditions; electrophoresing said gel matrix in a second dimension under a second set of electrophoresis conditions, such that linear nucleic acid fragments of equal length but having different conformation are separated; said first and second electrophoresis conditions are different, such that in one of said dimensions electrophoresis allows separation of linear nucleic acid fragments based on conformation and length, and in the other of said dimensions electrophoresis allows separation of the sample fragments based substantially on length. Said difference is preferably established with a chemical agent capable of reducing conformational differences between linear nucleic acids fragments.

Described is a process for the separation of a sample mixture for analytical reason based on two-dimensional gel electrophoresis. The method is involving a first separation in a first gel strip on the basis of isoelectric points and a second separation in a second gel on the basis of molecular size. When starting the separation in the second dimension the buffer solution for transferring the compounds separated in the first dimension into the second dimension gel is containing sodium dodecyl-sulfate (SDS) and by applying an electric field the SDS migrates electrokinetically into the first gel strip, and the compounds are being complexed simultaneously with SDS.

The present invention relates to a method for identification of cellular protein antigens to which patients with cancer, or patients at risk for cancer, may develop autoantibodies. The method of the invention involves the use of patient derived sera for the identification of the cellular protein antigens using two-dimensional gel electrophoresis followed by Western Blot analysis. The identification of such protein antigens provides novel markers that can be utilized for screening, for diagnostics and prognosis of disease. The invention also provides for the use of the identified protein antigens in immunoassays designed to detect the presence of serum antibodies to the specific protein antigens in sera from individuals that may harbor such antibodies. The invention further relates to the use of the identified antigens as immunogens for stimulation of an immune response in patients expressing such protein antigens. The invention is demonstrated by way of example in which elevated levels of circulating autoantibodies reactive against a tumor specific antigen were identified in sera derived from a lung cancer patient. In addition, elevated levels of circulating autoantibodies reactive against several specific beta-tubulin isoforms were detected in the sera of neuroblastoma patients.

This invention provides a method and usage for getting serum specificity protein for schizophrenia diagnosis, which utilizes a method of two-way gel electrophoresis to separate the under-test individual serum protein to get the expression volumes of alpha globin, alpha1 antitrypsin macroglobulin, serum amyloid p component, antifibrin-ferment III and vitamin D bindin to be taken as the reference basis for judging if a patient suffers from schizophrenia.

A method is provided for separating single- and double-stranded nucleic acid molecules based on their strandness and length. The method is based on novel two-dimensional gel electrophoresis techniques comprises loading a sample of nucleic acid molecules in a gel electrophoresis apparatus and electrophoresing in a first dimension said sample through a gel matrix under a first set of pre-determined electrophoresis conditions; electrophoresing said gel matrix in a second dimension under a second set of electrophoresis conditions, such that populations of single- and double-stranded nucleic acids are separated, said first and second electrophoresis conditions being different, such that in one dimension electrophoresis allows separation of the sample molecules based on strandness and length, and in the other dimension electrophoresis allows separation based substantially on length, wherein said difference is established with a chemical agent and / or physical parameter affecting the strandness-dependent electrophoresis migration rate of nucleic acids.

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.

Protein markers of toxicity and efficacy for antilipemic drugs are determined. Methods and reagents are disclosed for determining whether a patient receiving an antilipemic drug, especially a statin or HMGCoA reductase inhibiting drug, is experiencing drug efficacy and / or toxicity. Individual susceptibility is also determined prior to treatment. Also, drug discovery of similar acting candidates and their likelihood of being toxic or effective is determined by analysis of all proteins in a sample simultaneously by 2-dimensional gel electrophoresis.

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.

An electrophoresis assembly or disposable device for the separation of a complex protein sample, using two dimensional gel electrophoresis comprises a carrier such as a film or foil or glass plate (5) with an area for the first dimension gel strip (7) and an area (16) for the second dimension gel, where said two areas are directly in contact with each other. The assembly or device further comprises a two component main body made of a hard component (3) and a thin and flexible soft component layer (11) at a variable distance from the carrier (5) which body further comprises at least one slit or opening for external actuation of at least one valve (9, 10) where said valve is represented by said soft component material in correspondence of said at least one slit.

A method of predicting an effect of a test chemical on living organisms, which comprises:a step of administering a plurality of chemicals whose effects on living organisms are known, to respective chemical dosed groups, administering a test chemical to a test chemical dosed group, collecting proteins from each group after a definite period of time, separating the proteins by two-dimensional gel electrophoresis, measuring the signal intensities of spots of the proteins and modified proteins formed therefrom, and calculating a signal intensity ratio of at least two spots, anda step of comparing the signal intensity ratio of test chemical dosed group with the signal intensity ratio of each chemical dosed group.

A disposable electrophoresis device for separation of a complex protein sample, using two-dimensional gel electrophoresis and method thereof are disclosed. The device comprises a carrier with a first area for a first dimension gel strip and a second area for a second dimension gel, where the two areas are directly in contact with each other. The device further comprises a body made of a hard component and an elastic component layer at a variable distance from the carrier. The body further comprises at least one cavity for external actuation of at least one valve, wherein the valve is represented by the elastic component layer.

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 discloses a two-dimensional gel electrophoresis separation method of rice total protein under heavy metal stress. The method comprises the following steps: (a) extracting the coarse total protein of the rice root, washing with the mixed solution of ethyl ether and ethanol, performing freeze drying, dissolving in lysate, heating for 0.5-2 hours with a water bath, centrifuging, takingsupernatant to precipitate with acetone, centrifuging to separate the precipitate, and performing freeze drying; (b) dissolving the precipitate in thickened solution, precipitating with cold acetone under the temperature from -20 DEG C to -15 DEG C, centrifuging to separate the precipitate, repeating the step 2-4 times, performing freeze drying to the precipitate; and (c) dissolving the precipitate obtained in the step (b) in thickened solution, and separating by utilizing two-dimensional gel electrophoresis. In the method disclosed by the invention, surface detergents with different strengths such as urea and thiourea and various chaotropic agents are added, then polyvinyl pyrrolidone (PVP), ethanol and ethyl ether are added to wash the product and remove other impurities; and finally purer protein is obtained through stepwise dissolution, thus the complexity of the protein sample can be reduced and high purity total protein of the rice root can be obtained.

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 provides a goat milk proteome comparing method. The method includes the steps of a, collecting milk samples; b, preparing a solution; c, preparing skim milk protein samples; d, measuring the concentration of the sample protein, to be more specific, using a BCA kit, allowing PBS to be blank, using 5.0mg / mL BSA as the standard protein, and measuring the OD value of the sample protein with a microplate reader under the wavelength of 560 nanometers to obtain the protein concentration of the colostrum skim milk protein sample and the normal skim milk protein sample; e, performing two-dimensional gel electrophoresis, to be more specific, sequentially performing isoelectric focusing electrophoresis, gel strip equilibrium, SDS-PAGE vertical gel electrophoresis and gel image collecting and analysis to obtain the 2-DE spectrum of the colostrum milk sample and the normal milk sample. The goat milk proteome comparing method has the advantages that the protein components of biological systems can be analyzed in a large-scale manner at the same time, and a theoretical basis is provided for the systematic disclosing of the change law of goat milk proteome along with lactation stages.

This invention provides a method and usage for getting serum specificity protein for schizophrenia diagnosis, which utilizes a method of two-way gel electrophoresis to separate the under-test individual serum protein to get the expression volumes of alpha globin, alpha1 antitrypsin macroglobulin, serum amyloid p component, antifibrin-ferment III and vitamin D bindin to be taken as the reference basis for judging if a patient suffers from schizophrenia.

The invention provides a kidney yin deficiency syndrome animal model biomarker screening and determining method. The method comprises the following steps: extracting a cortical bone total protein sample, measuring the protein concentration, carrying out bidirectional gel electrophoresis analysis, detecting the gel protein spot, displaying and analyzing differential protein spots, carrying out MALDI-TOF-MS of differential protein spots, searching the protein database to identify and determine differential proteins, identifying part of differential proteins through Western-Bolt and RT-PCR, and interactively verifying the identification results with ELISA detection results of differential proteins in the serum. The differential proteins, whose expression trends are consistent and are not in the normal value range, can be used as the biomarker for identifying a kidney yin deficiency syndrome animal model. The biomarker can be used as the discrimination index of kidney yin deficiency syndrome in animal experiments; and a base is provided for finding another kidney yin deficiency syndrome biomarker.