Binding of Protein Factor CTCF within Chicken Genome Alpha-Globin Locus
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Abstract:
A systematic search for DNA fragments containing potential CTCF transcription factor binding sites in the chicken alpha-globin domain and its flanking regions was performed by means of the two-dimension electrophoretic mobility shift assay. For the alpha-globin domain fragments selected, the occupancy by the CTCF in erythroid and lymphoid chicken cells was tested by chromatin immunoprecipitation. Only one of 13 DNA fragments capable of CTCF binding in vitro was efficiently bound to this protein in vivo in erythroid cells, and somewhat less efficiently - in lymphoid cells. So, binding of CTCF to the DNA fragment in vitro in most cases does not mean that this fragment will be occupied by CTCF in the cell nucleus. Yet, CTCF binding in vivo, as a rule, is accompanied by the binding of the protein to this DNA region in vitro. During the erythroid differentiation, no significant changes in CTCF binding to the DNA fragments studied were detected.Keywords:
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Chromatin immunoprecipitation
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Abstract 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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Abstract In the past few decades, numerous approaches have been developed to investigate protein‐protein and protein‐nucleic acid interactions (PPIs and PNIs). Affinity purification methods such as co‐immunoprecipitation (co‐IP) are commonly used to detect and isolate the macromolecular complexesresulting from these interactions. In this article, we describe a two‐step co‐immunoprecipitation (TIP) technique. As compared to standard co‐IP, TIP provides increased specificity in the isolation of PPIs or PNIs under native expression conditions, dramatically reducing the abundance of nonspecific binders and thus facilitating downstream analyses of the interaction complexes. Here, we report a detailed TIP procedure that we used to purify a protein‐protein complex from Burkitt lymphoma cells and from primary human CD4 + T cells. In addition, this unit describes an application of TIP for the isolation of transcription‐factor‐bound chromatin. © 2018 by John Wiley & Sons, Inc.
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Chromatin immunoprecipitation
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This protocol can be ued for chromatin immunoprecipitation of RNAPII and associated factors, as well as histones. The settings are given for HeLa cells and should be adapted for other cell types.
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To verify the binding of p53 to p21WAF1/CIP1 gene promoter and detect its binding to hsp90 beta gene promoter in vivo.Chromatin immunoprecipitation and PCR analysis were used to measure specific gene regulation sequence and Western blot analysis to investigate p53 protein.The p53 binding sequences on the promoters of p21WAF1/CIP1 and hsp90 beta gene were found in the p53 antibody immunoprecipitated DNA fragments and p53 was detected in the immunoprecipitated samples.p53 binds to promoters of p21WAF1/CIP1 and hsp90 beta gene in vivo, and regulates the expression of the two genes.
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Chromatin immunoprecipitation (ChIP) assays are widely used to investigate where chromatin-binding proteins bind to the genome. The standard assay is very time consuming. We have developed a rapid ChIP assay in which the immunoprecipitates serve directly as PCR templates. This assay eliminates the step to reverse the crosslinking, shortening the assay by 1 day. It also requires a less immunoprecipitating antibody, permits many samples to be tested simultaneously, and is more sensitive than the standard ChIP assay.
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Chromatin immunoprecipitation
Immunoprecipitation
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Chromatin immunoprecipitation
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DNA-protein interactions are a major challenge in the research of gene transcription regulation.Chromatin immunoprecipitation assay(CHIP)provides a powerful tool to analyze this interaction in vivo.This article summarizes the methods of ChIP assay,and highlights the application and recent progress in the application of this technique.
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