Chromatin Immunoprecipitation (ChIP) UsingDrosophilaAntennal and Brain Samples
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Chromatin immunoprecipitation (ChIP) is a common approach for studying the binding pattern of proteins on DNA sequences or the landscape of histones with different marks throughout the genome. ChIP is used on various organisms, including Drosophila . This protocol provides a detailed overview of the immunoprecipitation portion of a ChIP procedure from samples of Drosophila nervous systems, specifically antennae and brains, that have already been fixed and sheared. These methods can be applied to other tissues of interest after optimizing for sample size and other relevant parameters.Keywords:
Chromatin immunoprecipitation
Immunoprecipitation
ChIP-on-chip
ChIP-sequencing
In the post-genomic era, identifying and characterzing various DNA-protein interactions are a major challenge in the research of gene transcriptional regulation. Now, chromatin immunoprecipitation assay (ChIP) is ideally suited for investigating DNA-protein interactions in vivo. The conditions of chromatin immunoprecipitation assay was optimized and utilized to screen unknown target genes of transcription factor AP-2 alpha. The results yielded a more complete understanding of AP-2α-regulated signaling pathways and further elucidated the complex transcriptional networks.
Chromatin immunoprecipitation
Immunoprecipitation
ChIP-on-chip
ChIA-PET
ChIP-sequencing
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Chromatin immunoprecipitation
Immunoprecipitation
ChIP-on-chip
ChIP-sequencing
Methylated DNA immunoprecipitation
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Chromatin immunoprecipitation (ChIP) is a widely used method to explore in vivo interactions between proteins and DNA. The ChIP assay takes several days to complete, involves several tube transfers and uses either phenol–chlorophorm or spin columns to purify DNA. The traditional ChIP method becomes a challenge when handling multiple samples. We have developed an efficient and rapid Chelex resin-based ChIP procedure that dramatically reduces time of the assay and uses only a single tube to isolate PCR-ready DNA. This method greatly facilitates the probing of chromatin changes over many time points with several antibodies in one experiment.
Chromatin immunoprecipitation
Immunoprecipitation
ChIP-sequencing
ChIP-on-chip
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Chromatin immunoprecipitation
Immunoprecipitation
ChIP-sequencing
ChIP-on-chip
ChIA-PET
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Immunoprecipitation
Chromatin immunoprecipitation
Nuclease
ChIP-on-chip
ChIP-sequencing
Micrococcal nuclease
ChIA-PET
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Immunoprecipitation
Chromatin immunoprecipitation
ChIP-on-chip
ChIP-sequencing
genomic DNA
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Chromatin immunoprecipitation
Immunoprecipitation
ChIP-sequencing
ChIP-on-chip
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Chromatin-immunoprecipitation (ChIP) is a powerful technique for mapping the protein-DNA interactions that occur in living cells. The critical technical determinant for successful ChIP is the availability of an appropriate, "ChIP-grade" serum. Here we present a technique designed to assess whether sera are suitable for ChIP, and to quantify their efficiency relative to a positive internal reference. This approach is useful as a first step toward ChIP-on-chip or ChIP-sequencing, especially in the case of recently identified proteins for which no binding sites are yet known.
Chromatin immunoprecipitation
Immunoprecipitation
ChIP-on-chip
ChIP-sequencing
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Chromatin immunoprecipitation (ChIP) is a technique widely used for determining the genomic location of modified histones and other chromatin-associated factors. Here we describe the methodology we have used in our laboratory for the immunoprecipitation of chromatin isolated from cells in the absence of crosslinking. Chromatin released from nuclei by micrococcal nuclease digestion is centrifuged through sucrose gradients to allow selection of monoor dinucleosomes. This allows a protein or modification at a particular gene or locus to be mapped at higher resolution than in a crosslinked ChIP experiment. Two methods for the immunoprecipitation of chromatin are described: a large-scale fractionation by which it is possible to visualize the proteins of the immunoprecipitate by polyacrylamide gel electrophoresis, PAGE and a small-scale method that is more appropriate when the quantity of chromatin is limited. The sequence content of DNA extracted from the immunoprecipitated chromatin is analyzed by hybridization of Southern or slot blots, or by quantitative polymerase chain reaction. Enrichment of particular sequences in the immunoprecipitated fraction reveals the presence and extent of the modification at this location.
Chromatin immunoprecipitation
Immunoprecipitation
Micrococcal nuclease
ChIP-sequencing
ChIP-on-chip
Nuclease
ChIA-PET
Cite
Citations (35)
Chromatin immunoprecipitation (ChIP) is a common approach for studying the binding pattern of proteins on DNA sequences or the landscape of histones with different marks throughout the genome. ChIP is used on various organisms, including Drosophila . This protocol provides a detailed overview of the immunoprecipitation portion of a ChIP procedure from samples of Drosophila nervous systems, specifically antennae and brains, that have already been fixed and sheared. These methods can be applied to other tissues of interest after optimizing for sample size and other relevant parameters.
Chromatin immunoprecipitation
Immunoprecipitation
ChIP-on-chip
ChIP-sequencing
Cite
Citations (0)