Protocol for the fast chromatin immunoprecipitation (ChIP) method

Nelson, Joel D.; Denisenko, Oleg; Bomsztyk, Karol

doi:10.1038/nprot.2006.27

Cited by 702 publications

(573 citation statements)

References 28 publications

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“…Here we tested an extraction buffer containing 150 mmol L 1 NaCl, 0.1% Triton X-100, and protease inhibitor cocktail. This buffer also provides a more stringent environment for protein-protein interactions, which will increase the antibody binding specificity and signal-to-noise ratio in downstream experiments like native chromatin immunoprecipitation (N-ChIP) (Nelson et al, 2006). Our result showed that the extraction buffer produced a dramatic increase in the yield of mono-nucleosomes, and even more so, di-nucleosomes ( Figure 3D).…”

Section: Optimization Of Mnase Digestion For Macsupporting

confidence: 49%

Enzymatic and chemical mapping of nucleosome distribution in purified micro- and macronuclei of the ciliated model organism, Tetrahymena thermophila

Chen

Gao

Liu

et al. 2016

Sci. China Life Sci.

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Genomic distribution of the nucleosome, the basic unit of chromatin, contains important epigenetic information. To map nucleosome distribution in structurally and functionally differentiated micronucleus (MIC) and macronucleus (MAC) of the ciliate Tetrahymena thermophila, we have purified MIC and MAC and performed micrococcal nuclease (MNase) digestion as well as hydroxyl radical cleavage. Different factors that may affect MNase digestion were examined, to optimize mono-nucleosome production. Mono-nucleosome purity was further improved by ultracentrifugation in a sucrose gradient. As MNase concentration increased, nucleosomal DNA sizes in MIC and MAC converged on 147 bp, as expected for the nucleosome core particle. Both MNase digestion and hydroxyl radical cleavage consistently showed a nucleosome repeat length of ~200 bp in MAC of Tetrahymena, supporting ~50 bp of linker DNA. Our work has systematically tested methods currently available for mapping nucleosome distribution in Tetrahymena, and provided a solid foundation for future epigenetic studies in this ciliated model organism.

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Section: Optimization Of Mnase Digestion For Macsupporting

confidence: 49%

Enzymatic and chemical mapping of nucleosome distribution in purified micro- and macronuclei of the ciliated model organism, Tetrahymena thermophila

Chen

Gao

Liu

et al. 2016

Sci. China Life Sci.

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“…Chromatin Immunoprecipitation (ChIP) Assay-ChIP assay was performed as described previously (22). Anti-NF-YA or normal mouse IgG (Santa Cruz Biotechnology) antibodies were used in immunoprecipitation experiments.…”

Section: Establishment Of Tumors and Dna Delivery-n-[1-(23-dio-mentioning

confidence: 99%

Transcriptional Activation of Human CDCA8 Gene Regulated by Transcription Factor NF-Y in Embryonic Stem Cells and Cancer Cells

Dai

Miao

et al. 2015

Journal of Biological Chemistry

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Background:The regulation of CDCA8 gene expression remains unclear. Results: CDCA8 promoter is activated in hESCs and cancer cells, in which NF-Y is a positive-regulator by binding to the CCAAT-box. Conclusion: CDCA8 is transcriptionally activated in hESCs and cancer cells and is positively regulated by NF-Y. Significance: Characterization of CDCA8 regulation provides a better understanding of its biological functions.

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“…ChIP analysis of in vivo binding of p53 to target promoters was performed as described (Nelson et al 2006). Briefly, cells were fixed with 1.42% formaldehyde for 15 min at room temperature, then neutralized with 125 mM glycine for 5 min.…”

Section: Subcellular Fractionationmentioning

confidence: 99%

Δ40p53 controls the switch from pluripotency to differentiation by regulating IGF signaling in ESCs

Ungewitter¹,

Scrable²

2010

Genes Dev.

113

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D40p53 is a transactivation-deficient isoform of the tumor suppressor p53. We discovered that D40p53, in addition to being highly expressed in embryonic stem cells (ESCs), is the major p53 isoform during early stages of embryogenesis in the mouse. By altering the dose of D40p53 in ESCs, we identified a critical role for this isoform in maintaining the ESC state. Haploinsufficiency for D40p53 causes a loss of pluripotency in ESCs and acquisition of a somatic cell cycle, while increased dosage of D40p53 prolongs pluripotency and inhibits progression to a more differentiated state. D40p53 controls the switch from pluripotent ESCs to differentiated somatic cells by controlling the activity of full-length p53 at critical targets such as Nanog and the IGF-1 receptor (IGF-1R). The IGF axis plays a central role in the switch between pluripotency and differentiation in ESCs-and D40p53, by controlling the level of the IGF-1R, acts as a master regulator of this switch. We propose that this is the primary function of D40p53 in cells of the early embryo and stem cells, which are the only normal cells in which this isoform is expressed.[Keywords: D40p53; embryonic stem cells; pluripotency; IGF] Supplemental material is available at http://www.genesdev.org.

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Protocol for the fast chromatin immunoprecipitation (ChIP) method

Cited by 702 publications

References 28 publications

Enzymatic and chemical mapping of nucleosome distribution in purified micro- and macronuclei of the ciliated model organism, Tetrahymena thermophila

Enzymatic and chemical mapping of nucleosome distribution in purified micro- and macronuclei of the ciliated model organism, Tetrahymena thermophila

Transcriptional Activation of Human CDCA8 Gene Regulated by Transcription Factor NF-Y in Embryonic Stem Cells and Cancer Cells

Δ40p53 controls the switch from pluripotency to differentiation by regulating IGF signaling in ESCs

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