Assessment of sera for chromatin-immunoprecipitation
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Abstract:
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.Keywords:
Chromatin immunoprecipitation
Immunoprecipitation
ChIP-on-chip
ChIP-sequencing
Chromatin immunoprecipitation
ChIP-sequencing
ChIP-on-chip
Immunoprecipitation
genomic DNA
ChIA-PET
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Abstract Chromatin immunoprecipitation (ChIP) is a powerful and widely applied technique for detecting the association of individual proteins with specific genomic regions in vivo. Live cells are treated with formaldehyde to generate protein‐protein and protein‐DNA cross‐links between molecules that are in close proximity on the chromatin template in vivo. DNA sequences that cross‐link with a given protein are selectively enriched, and reversal of the formaldehyde cross‐linking permits recovery and quantitative analysis of the immunoprecipitated DNA. As formaldehyde inactivates cellular enzymes essentially immediately upon addition to cells, ChIP provides snapshots of protein‐protein and protein‐DNA interactions at a particular time point, and hence is useful for kinetic analysis of events occurring on chromosomal sequences in vivo. In addition, ChIP can be combined with microarray technology to identify the location of specific proteins on a genome‐wide basis. in this unit describes the ChIP procedure for Saccharomyces cerevisiae ; describes the corresponding steps for mammalian cells.
Chromatin immunoprecipitation
ChIP-on-chip
Immunoprecipitation
ChIP-sequencing
genomic DNA
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Chromatin immunoprecipitation (ChIP) is a powerful tool to study protein-DNA interaction and is widely used in many fields to study proteins associated with chromatin, such as histone and its isoforms and transcription factors, across a defined DNA domain. Here, we show the step-by-step methods currently used in our lab to immunoprecipitate the formaldehyde crosslinked chromatin and further analyze the immuprecipitated DNA by semiquantitative PCR.
Chromatin immunoprecipitation
ChIP-on-chip
ChIP-sequencing
Immunoprecipitation
ChIA-PET
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Chromatin immunoprecipitation
ChIP-on-chip
ChIP-sequencing
Immunoprecipitation
ChIA-PET
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Immunoprecipitation
Chromatin immunoprecipitation
Nuclease
ChIP-on-chip
ChIP-sequencing
Micrococcal nuclease
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Immunoprecipitation
Chromatin immunoprecipitation
ChIP-on-chip
ChIP-sequencing
genomic DNA
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Expression of eukaryotic genes during development requires complex spatial-temporal regulation. This complex regulation is often achieved through the coordinated interaction of transcription regulatory elements in the promoters of the target genes. The identification and mapping of regulatory elements in genome scale is crucial to understand how gene expression is regulated. Chromatin immunoprecipitation is a standard method for assessing the occupancy of DNA binding proteins in vivo in their native chromatin context using antibodies. However, standard chromatin immunoprecipitation procedure is time consuming, labor intensive and not suited for analyzing many samples simultaneously.
Recently, we have developed a simple ChIP protocol that requires fewer steps and less hands-on time. This protocol is compatible with both 96-well plate and single tube formats, and enables higher sensitivity and more reliable performance, as compared to conventional approaches.
We have successfully used this protocol to map various clinically relevant chromatin marks and controls across several cell types to quantitatively measure chromatin states. This analysis included a variety of marks corresponding to repressed, poised and active promoters, strong and weak enhancers, putative insulators, transcribed regions, as well as large-scale repressed and inactive domains. This study demonstrates the utility of this approach for the characterization of model cellular systems in perturbation studies with chemical probes.
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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
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In the post-genomic era, identifying and characterizing various DNA-protein interactions are a major challenge in the research of gene transcriptional regulation. Although many techniques can be used for this purpose, chromatin immunoprecipitation assay (ChIP), by contrast, is ideally suited for studying DNA-protein interactions in vivo. In recent years, standard ChIP assay has been modified to uncover some known factors' unknown target sequences, especially when combined with DNA microarray and molecular cloning strategies. These high-throughput ChIP assays are more and more used to reveal the distribution profile of trans-acting factor binding sites throughout the genome, which may yield many new insights into the DNA-protein interaction network. This article summarized the methods of ChIP assay, and highlighted recent progress in the application of this technique.
Chromatin immunoprecipitation
ChIP-sequencing
ChIP-on-chip
Immunoprecipitation
ChIA-PET
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Chromatin immunoprecipitation
Immunoprecipitation
ChIP-on-chip
ChIP-sequencing
ChIA-PET
Cite
Citations (38)