The Experts below are selected from a list of 321 Experts worldwide ranked by ideXlab platform
Kevin Struhl - One of the best experts on this subject based on the ideXlab platform.
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Analysis of protein co-occupancy by quantitative sequential chromatin Immunoprecipitation.
Current protocols in molecular biology, 2005Co-Authors: Joseph V. Geisberg, Kevin StruhlAbstract:Sequential Chromatin Immunoprecipitation (SeqChIP) is a powerful technique for analyzing the simultaneous association of two different proteins with genomic DNA sequences in vivo. Cellular Protein-DNA complexes are cross-linked with formaldehyde (UNIT ), and are purified via two successive Immunoprecipitations, with each Immunoprecipitation targeting a different protein. Protein-DNA cross-links are then reversed and DNA sequences of interest are analyzed by quantitative PCR. At each genomic region, calculated SeqChIP co-occupancy values are compared to occupancy values of singly immunoprecipitated samples. The extent of enrichment brought about by the second Immunoprecipitation relative to the singly immunoprecipitated sample is directly correlated with the degree of co-occupancy between the two proteins at the genomic location assayed. In principle, the technique is not limited to Saccharomyces cerevisiae. Cells from a wide variety of organisms can be used.
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UNIT 21.8 Analysis of Protein Co-Occupancy by Quantitative Sequential Chromatin Immunoprecipitation
Current Protocols in Molecular Biology, 2004Co-Authors: Joseph V. Geisberg, Kevin StruhlAbstract:Sequential Chromatin Immunoprecipitation (SeqChIP) is a powerful technique for analyzing the simultaneous association of two different proteins with genomic DNA sequences in vivo. Cellular Protein-DNA complexes are cross-linked with formaldehyde (unit Unavailable ), and are purified via two successive Immunoprecipitations, with each Immunoprecipitation targeting a different protein. Protein-DNA cross-links are then reversed and DNA sequences of interest are analyzed by quantitative PCR. At each genomic region, calculated SeqChIP co-occupancy values are compared to occupancy values of singly immunoprecipitated samples. The extent of enrichment brought about by the second Immunoprecipitation relative to the singly immunoprecipitated sample is directly correlated with the degree of co-occupancy between the two proteins at the genomic location assayed. In principle, the technique is not limited to Saccharomyces cerevisiae. Cells from a wide variety of organisms can be used. Keywords: chromatin; Immunoprecipitation; protein-DNA interactions; in vivo crosslinking
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quantitative sequential chromatin Immunoprecipitation a method for analyzing co occupancy of proteins at genomic regions in vivo
Nucleic Acids Research, 2004Co-Authors: Joseph V. Geisberg, Kevin StruhlAbstract:Sequential chromatin Immunoprecipitation (SeqChIP) is a procedure in which formaldehyde-crosslinked, protein–DNA complexes from living cells are subjected to two sequential Immunoprecipitations with antibodies of different specificity. SeqChIP has been used to address, in a qualitative manner, whether two proteins can simultaneously co-occupy a stretch of DNA in vivo. Here, we expand on our earlier work and describe theoretical and practical considerations for performing and interpreting SeqChIP experiments in a quantitative manner. We provide a detailed experimental procedure for designing and performing SeqChIP experiments as well as experimental examples of the three possible outcomes: full co-occupancy, no co-occupancy and partial co-occupancy. In some cases of partial co-occupancy, the order of Immunoprecipitations in SeqChIP can strongly influence the outcome. We experimentally confirm a quantitative parameter that provides a measure of co-occupancy of two proteins on a given region of DNA and provide information on how to interpret the results of SeqChIP experiments. Our quantitative treatment of SeqChIP data substantially expands the usefulness of the technique for elucidating molecular mechanisms in vivo.
Joseph V. Geisberg - One of the best experts on this subject based on the ideXlab platform.
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Analysis of protein co-occupancy by quantitative sequential chromatin Immunoprecipitation.
Current protocols in molecular biology, 2005Co-Authors: Joseph V. Geisberg, Kevin StruhlAbstract:Sequential Chromatin Immunoprecipitation (SeqChIP) is a powerful technique for analyzing the simultaneous association of two different proteins with genomic DNA sequences in vivo. Cellular Protein-DNA complexes are cross-linked with formaldehyde (UNIT ), and are purified via two successive Immunoprecipitations, with each Immunoprecipitation targeting a different protein. Protein-DNA cross-links are then reversed and DNA sequences of interest are analyzed by quantitative PCR. At each genomic region, calculated SeqChIP co-occupancy values are compared to occupancy values of singly immunoprecipitated samples. The extent of enrichment brought about by the second Immunoprecipitation relative to the singly immunoprecipitated sample is directly correlated with the degree of co-occupancy between the two proteins at the genomic location assayed. In principle, the technique is not limited to Saccharomyces cerevisiae. Cells from a wide variety of organisms can be used.
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UNIT 21.8 Analysis of Protein Co-Occupancy by Quantitative Sequential Chromatin Immunoprecipitation
Current Protocols in Molecular Biology, 2004Co-Authors: Joseph V. Geisberg, Kevin StruhlAbstract:Sequential Chromatin Immunoprecipitation (SeqChIP) is a powerful technique for analyzing the simultaneous association of two different proteins with genomic DNA sequences in vivo. Cellular Protein-DNA complexes are cross-linked with formaldehyde (unit Unavailable ), and are purified via two successive Immunoprecipitations, with each Immunoprecipitation targeting a different protein. Protein-DNA cross-links are then reversed and DNA sequences of interest are analyzed by quantitative PCR. At each genomic region, calculated SeqChIP co-occupancy values are compared to occupancy values of singly immunoprecipitated samples. The extent of enrichment brought about by the second Immunoprecipitation relative to the singly immunoprecipitated sample is directly correlated with the degree of co-occupancy between the two proteins at the genomic location assayed. In principle, the technique is not limited to Saccharomyces cerevisiae. Cells from a wide variety of organisms can be used. Keywords: chromatin; Immunoprecipitation; protein-DNA interactions; in vivo crosslinking
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quantitative sequential chromatin Immunoprecipitation a method for analyzing co occupancy of proteins at genomic regions in vivo
Nucleic Acids Research, 2004Co-Authors: Joseph V. Geisberg, Kevin StruhlAbstract:Sequential chromatin Immunoprecipitation (SeqChIP) is a procedure in which formaldehyde-crosslinked, protein–DNA complexes from living cells are subjected to two sequential Immunoprecipitations with antibodies of different specificity. SeqChIP has been used to address, in a qualitative manner, whether two proteins can simultaneously co-occupy a stretch of DNA in vivo. Here, we expand on our earlier work and describe theoretical and practical considerations for performing and interpreting SeqChIP experiments in a quantitative manner. We provide a detailed experimental procedure for designing and performing SeqChIP experiments as well as experimental examples of the three possible outcomes: full co-occupancy, no co-occupancy and partial co-occupancy. In some cases of partial co-occupancy, the order of Immunoprecipitations in SeqChIP can strongly influence the outcome. We experimentally confirm a quantitative parameter that provides a measure of co-occupancy of two proteins on a given region of DNA and provide information on how to interpret the results of SeqChIP experiments. Our quantitative treatment of SeqChIP data substantially expands the usefulness of the technique for elucidating molecular mechanisms in vivo.
Jurgen Gotz - One of the best experts on this subject based on the ideXlab platform.
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co Immunoprecipitation with tau isoform specific antibodies reveals distinct protein interactions and highlights a putative role for 2n tau in disease
Journal of Biological Chemistry, 2016Co-Authors: Chang Liu, Xiaomin Song, Rebecca M Nisbet, Jurgen GotzAbstract:Alternative splicing generates multiple isoforms of the microtubule-associated protein Tau, but little is known about their specific function. In the adult mouse brain, three Tau isoforms are expressed that contain either 0, 1, or 2 N-terminal inserts (0N, 1N, and 2N). We generated Tau isoform-specific antibodies and performed co-Immunoprecipitations followed by tandem mass tag multiplexed quantitative mass spectrometry. We identified novel Tau-interacting proteins of which one-half comprised membrane-bound proteins, localized to the plasma membrane, mitochondria, and other organelles. Tau was also found to interact with proteins involved in presynaptic signal transduction. MetaCore analysis revealed one major Tau interaction cluster that contained 33 Tau pulldown proteins. To explore the pathways in which these proteins are involved, we conducted an ingenuity pathway analysis that revealed two significant overlapping pathways, "cell-to-cell signaling and interaction" and "neurological disease." The functional enrichment tool DAVID showed that in particular the 2N Tau-interacting proteins were specifically associated with neurological disease. Finally, for a subset of Tau interactions (apolipoprotein A1 (apoA1), apoE, mitochondrial creatine kinase U-type, β-synuclein, synaptogyrin-3, synaptophysin, syntaxin 1B, synaptotagmin, and synapsin 1), we performed reverse co-Immunoprecipitations, confirming the preferential interaction of specific isoforms. For example, apoA1 displayed a 5-fold preference for the interaction with 2N, whereas β-synuclein showed preference for 0N. Remarkably, a reverse Immunoprecipitation with apoA1 detected only the 2N isoform. This highlights distinct protein interactions of the different Tau isoforms, suggesting that they execute different functions in brain tissue.
Neil Mchugh - One of the best experts on this subject based on the ideXlab platform.
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anti synthetase syndrome a new autoantibody to phenylalanyl transfer rna synthetase anti zo associated with polymyositis and interstitial pneumonia
Rheumatology, 2007Co-Authors: Zoe Betteridge, Harsha Gunawardena, J North, Jenna Slinn, Neil MchughAbstract:Objective. Autoantibodies directed against the aminoacyl tRNA synthetases are associated with myositis, arthritis, Raynaud’s phenomenon, mechanic’s hands, fever and interstitial pneumonia, clinically referred to as the anti-synthetase syndrome (ASS). The aim of this study was to characterize the autoantibody profile in a patient with clinical features of ASS whose routine diagnostic testing was negative for the previously identified anti-synthetase autoantibodies. Methods. Serum from a patient presenting with interstitial pneumonia followed by proximal myopathy, Raynaud’s phenomenon and arthrlagia was analysed for autoantigen specificity by routine methods including indirect immunofluorescence, immunodiffusion, ELISA and immunoblotting. The autoantibody specificity was further analysed by RNA and protein Immunoprecipitation. Novel autoantigens found on protein Immunoprecipitation were further characterized using a proteomic approach, combining Immunoprecipitation, SDS-PAGE and MALDI-TOF mass spectrometry. Results. Diagnostic testing on the patient’s serum was negative by ELISA and immunodiffusion. Indirect immunofluorescence using Hep-2 cells was ANA negative, although a strong cytoplasmic speckle was seen. Immunoblotting with the patient serum displayed an unknown positive band at approximately 60kDa. Protein Immunoprecipitation revealed the presence of two proteins with molecular weights of approximately 60 and 70kDa, and RNA Immunoprecipitation revealed the presence of a band corresponding to a tRNA synthetase. Using a combination of Immunoprecipitation and mass spectrometry, the novel Immunoprecipitation targets were identified as phenylalanyl tRNA synthetase alpha and beta chains. Conclusions. We report the identification of previously uncharacterized autoantibodies to phenylalanyl tRNA synthetase, entitled anti-Zo. This is the eighth anti-synthetase autoantibody in a patient with anti-synthetase syndrome.
Chang Liu - One of the best experts on this subject based on the ideXlab platform.
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co Immunoprecipitation with tau isoform specific antibodies reveals distinct protein interactions and highlights a putative role for 2n tau in disease
Journal of Biological Chemistry, 2016Co-Authors: Chang Liu, Xiaomin Song, Rebecca M Nisbet, Jurgen GotzAbstract:Alternative splicing generates multiple isoforms of the microtubule-associated protein Tau, but little is known about their specific function. In the adult mouse brain, three Tau isoforms are expressed that contain either 0, 1, or 2 N-terminal inserts (0N, 1N, and 2N). We generated Tau isoform-specific antibodies and performed co-Immunoprecipitations followed by tandem mass tag multiplexed quantitative mass spectrometry. We identified novel Tau-interacting proteins of which one-half comprised membrane-bound proteins, localized to the plasma membrane, mitochondria, and other organelles. Tau was also found to interact with proteins involved in presynaptic signal transduction. MetaCore analysis revealed one major Tau interaction cluster that contained 33 Tau pulldown proteins. To explore the pathways in which these proteins are involved, we conducted an ingenuity pathway analysis that revealed two significant overlapping pathways, "cell-to-cell signaling and interaction" and "neurological disease." The functional enrichment tool DAVID showed that in particular the 2N Tau-interacting proteins were specifically associated with neurological disease. Finally, for a subset of Tau interactions (apolipoprotein A1 (apoA1), apoE, mitochondrial creatine kinase U-type, β-synuclein, synaptogyrin-3, synaptophysin, syntaxin 1B, synaptotagmin, and synapsin 1), we performed reverse co-Immunoprecipitations, confirming the preferential interaction of specific isoforms. For example, apoA1 displayed a 5-fold preference for the interaction with 2N, whereas β-synuclein showed preference for 0N. Remarkably, a reverse Immunoprecipitation with apoA1 detected only the 2N isoform. This highlights distinct protein interactions of the different Tau isoforms, suggesting that they execute different functions in brain tissue.
