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ChromatinChromatin ImmunoprecipitationChromatin Assembly and DisassemblyPromoter Regions, GeneticHistonesTranscription, GeneticTranscription FactorsProtein BindingAcetylationImmunoprecipitationGene Expression RegulationDNA-Binding ProteinsResponse ElementsBinding SitesNucleosomesBase SequencePrecipitin TestsCell LineTranscriptional ActivationCell Line, TumorRepressor ProteinsNuclear ProteinsMolecular Sequence DataElectrophoretic Mobility Shift AssayGene SilencingHistone DeacetylasesChromosomal Proteins, Non-HistoneSp1 Transcription FactorDNACell NucleusEpigenesis, GeneticRNA, MessengerRNA, Small InterferingMethylationCells, CulturedTrans-ActivatorsHistone AcetyltransferasesHeLa CellsRNA Polymerase IIDNA MethylationReverse Transcriptase Polymerase Chain ReactionBlotting, WesternMicrococcal NucleaseTransfectionRNA InterferenceGene Expression Regulation, NeoplasticMutationGenes, ReporterOligonucleotide Array Sequence AnalysisHydroxamic AcidsSaccharomyces cerevisiae ProteinsHeterochromatinp300-CBP Transcription FactorsSignal TransductionGene Knockdown TechniquesLysineLuciferasesTranscription Initiation SiteHistone-Lysine N-MethyltransferaseSaccharomyces cerevisiaeEnhancer Elements, GeneticHistone Deacetylase 1PhosphorylationCCAAT-Binding FactorHistone Deacetylase InhibitorsCell Cycle ProteinsGene Expression Regulation, DevelopmentalUp-RegulationCell DifferentiationProtein Structure, TertiaryDown-RegulationDNA PrimersDeoxyribonuclease IHomeodomain ProteinsAmino Acid SequenceGene Expression ProfilingE1A-Associated p300 ProteinRegulatory Elements, TranscriptionalGene Expression Regulation, EnzymologicGene Expression Regulation, FungalE-Box ElementsModels, GeneticModels, BiologicalKruppel-Like Transcription FactorsNIH 3T3 CellsChromatin Assembly Factor-1CentromereSex ChromatinEuchromatinCpG IslandsSp3 Transcription FactorAcetyltransferasesProtein Processing, Post-TranslationalCREB-Binding ProteinPlasmids5' Flanking RegionCell CycleGenomeGene ExpressionCell Proliferation
AssayChIP-SeqAssaysHistonesNative chromatinGuided by chromatin structureSequencingMicrococcal nuclease digestionNucleosomesProteins and chromatinMass spectrometryParticular chromatinHistone modificationsModificationsGenesReagentsProteinSonicationAntibodyDigestionCellsChromosomeShearModifiersExperimentsPreparationRegulationPromoterStructureTranscription factorBindingGene expressionWidelyExperimental techniqueSolutionProfilesCross-linked
Assay9
  • ATAC-seq(Assay for Transposase-Accessible Chromatin using sequencing)は、ゲノムワイドなクロマチンアクセシビリティの査定を可能にします。 この技術は、トランスポゼー (diagenode.com)
  • For this study, we are discussing new challenges in using chromatin immunoprecipitation (ChIP) assay to confirm protein - DNA interactions and identifying interacting proteins by mass spectrometry. (okstate.edu)
  • Method: The key component of the chromatin immunoprecipitation assay is the size of the DNA, which needs to be fragmented in a reliable consistent and repeatable manner. (okstate.edu)
  • Here, we used a chromatin immunoprecipitation assay to study the molecular mechanism responsible for this expression pattern. (aacrjournals.org)
  • To date, the most widely used and powerful method to identify regions of the genome associated with specific proteins is the Chromatin Immunoprecipitation (ChIP) assay. (thermofisher.com)
  • Expression of SIRT1/SOX9/LCN2 in the hypoxic cells was determined, and their interactions were predicted by bioinformatics websites and followed by the verification by luciferase assay and chromatin immunoprecipitation (ChIP). (hindawi.com)
  • Using a CRISPR Cas9-guided immunoprecipitation assay, we identify a circular RNA in the FLI1 promoter chromatin complex, consisting of FLI1 exons 4-2-3, referred to as FECR1.Overexpression of FECR1 enhances invasiveness of MDA-MB231 breast cancer cells. (biomedcentral.com)
  • In this study, we harnessed a novel CRISPR Cas9-guided promoter immunoprecipitation (CasIP) assay to identify the molecular components that interact with the FLI1 promoter. (biomedcentral.com)
  • Ketamine treatment increased the binding of NF‑κB and permissive histone H3 lysine‑4 (H3K4)m3, but caused a decrease in the repressive histone H3K27m3 and H3K36m3 on the COX‑2 promoter ranging from ‑1,522 to ‑1,331 bp as determined by a chromatin immunoprecipitation assay. (spandidos-publications.com)
ChIP-Seq6
  • Chromatin Immunoprecipitation sequencing, also known as ChIP-seq, is an experimental technique used to identify transcription factor binding events throughout an entire genome. (wikipedia.org)
  • It is optimized for ChIP reactions with chromatin from 10 6 cells (up to ~50 μg DNA), and can also be scaled up (or several preparations pooled) to accommodate 10 8 cells for genome-wide binding studies in ChIP-chip and ChIP-Seq applications. (sigmaaldrich.com)
  • Employs DNA-Blocked 'Staph-Seq' for IP (immunoprecipitation), minimizing contaminating Staph A DNA in downstream ChIP-Seq applications. (sigmaaldrich.com)
  • Purification of nanogram-range immunoprecipitated DNA in ChIP-seq utility Chromatin immunoprecipitation-sequencing (ChIP-seq) is a broadly used epigenetic strategy for investigating genome-wide protein-DNA interactions in cells and tissues. (chipready.com)
  • We previously developed a modified ChIP-seq protocol (FiT-seq) for chromatin profiling in FFPE. (nih.gov)
  • We performed chromatin immunoprecipitation sequencing (ChIP-seq) using a histone 3 lysine 9 acetylation (H3K9ac) mark in peripheral blood mononuclear cells from patients with atherosclerosis with T2D (n = 8) or without T2D (ND, n = 10). (lu.se)
Assays4
Histones5
Native chromatin7
Guided by chromatin structure1
  • One pathway, which has been extensively studied in yeast, is mainly guided by chromatin structure and the other, analyzed in detail in mice, is driven by the sequence-specific DNA-binding PR domain-containing protein 9 (PRDM9). (springer.com)
Sequencing3
Micrococcal nuclease digestion2
Nucleosomes2
  • Nucleosomes, the fundamental constructions used to bundle genetic info into chromatin, are topic to a various array of chemical modifications. (chipready.com)
  • The EZ-Zyme Chromatin Prep kit allows ChIP analysis at nucleosome resolution by performing complete or partial digestions with a proprietary enzymatic cocktail to obtain chromatin fragments of on average one to a few nucleosomes in length. (sigmaaldrich.com)
Proteins and chromatin1
  • ChIP can be used to answer a multitude of scientific questions involving the interaction of proteins and chromatin. (cellsignal.com)
Mass spectrometry2
  • Rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) is a method that allows the study of protein complexes, in particular chromatin and transcription factor complexes, in a rapid and robust manner by mass spectrometry (MS). The method can be used in parallel with chromatin immunoprecipitation-sequencing (ChIP-seq) experiments to provide information on both the cistrome and interactome for a given protein. (nature.com)
  • Aim: In this current study, we are identifying the challenges in optimizing chromatin immunoprecipitation and proteomics/mass spectrometry. (okstate.edu)
Particular chromatin1
  • ChIP has also been used to determine the temporal regulation underlying the occupation of the particular chromatin locus by multiple proteins. (sigmaaldrich.com)
Histone modifications2
  • Although it is clear that switching among BESs does not involve DNA rearrangements and that regulation is probably epigenetic, it remains unknown why BESs are transcribed by Pol I and what roles are played by chromatin structure and histone modifications. (asm.org)
  • Studies of genes transcribed by RNA polymerase II (Pol II) have shown that open and closed chromatin structures are dynamically regulated through multiple mechanisms, including histone modifications, histone variant incorporation, and DNA methylation (reviewed in reference 17 ). (asm.org)
Modifications1
  • The phytochemical 3,3'-diindolylmethane decreases expression of AR-controlled DNA damage repair genes through repressive chromatin modifications and is associated with DNA damage in prostate cancer cells. (oregonstate.edu)
Genes1
Reagents3
Protein13
  • The DNA-protein complexes (chromatin-protein) are then sheared into ~500 bp DNA fragments by sonication or nuclease digestion. (wikipedia.org)
  • The immunoprecipitated complexes (i.e., the bead-antibody-protein-target DNA sequence complex) are then collected and washed to remove non-specifically bound chromatin, the protein-DNA cross-link is reversed and proteins are removed by digestion with proteinase K. An epitope-tagged version of the protein of interest, or in vivo biotinylation can be used instead of antibodies to the native protein of interest. (wikipedia.org)
  • The principle behind ChIP is relatively straightforward and relies on the use of an antibody to isolate, or precipitate, a certain protein, histone, transcription factor, or cofactor and its bound chromatin from a protein mixture that was extracted from cells or tissues. (cellsignal.com)
  • In addition, loss of protein binding during the chromatin digestion and immunoprecipitation steps may bias the data or impede proper analyses. (cellsignal.com)
  • In X-ChIP, chemical fixatives such as formaldehyde are used to crosslink the protein of interest to the DNA and fragmentation of chromatin is achieved through sonication or nuclease digestion. (cellsignal.com)
  • X-ChIP also minimizes the chances of chromatin protein loss during extraction, allowing for the detection of transient protein interactions. (cellsignal.com)
  • It uses discs of an inert, porous, polymer to which Protein A or Protein G has been covalently attached to maximize the capture efficiency of the target chromatin/antibody complex. (technologynetworks.com)
  • Importantly, antibodies that recognize a protein of interest can be used to determine the relative association of that antigen in the context of chromatin at one or more loci in the genome. (thermofisher.com)
  • A key aspect for a successful ChIP is having antibodies that recognize the target protein in the context of chromatin. (thermofisher.com)
  • Key elements of a conventional protocol use an overnight immunoprecipitation step of antibody and chromatin, followed by enrichment with Protein A sepharose beads and extensive washes with buffers containing variable salt concentrations. (thermofisher.com)
  • When performing ChIP, chromatin from cells and tissues needs to be fragmented so that it becomes soluble and resolution can be achieved in detecting protein-DNA interaction at specific loci. (sigmaaldrich.com)
  • For example, foaming and overheating associated with sonication can result in protein denaturation or incomplete chromatin fragmentation. (sigmaaldrich.com)
  • Compared to sonication, this procedure not only allows mapping the protein-DNA association at a higher resolution, but also renders the subsequent immunoprecipitation more efficient due to its mild digestion condition. (sigmaaldrich.com)
Sonication4
Antibody3
Digestion1
  • Mild formaldehyde crosslinking followed by nuclease digestion has been used to shear the chromatin. (wikipedia.org)
Cells3
  • Briefly, the conventional method is as follows: DNA and associated proteins on chromatin in living cells or tissues are crosslinked (this step is omitted in Native ChIP). (wikipedia.org)
  • Sheared chromatin from MCF7 cells was prepared from 1 million cells in 50 μl lysis buffer and diluted to indicated number of cells per ChIP according to the MAGnify™ ChIP protocol. (thermofisher.com)
  • Sheared chromatin from 293Gt cells was prepared and 50,000 cells used for ChIP (MAGnify™ ChIP) and 1 million cells per ChIP used for the conventional ChIP protocol. (thermofisher.com)
Chromosome3
  • Chromosome construction seize expertise can detect the three-dimensional building of chromatin. (chipready.com)
  • 2009 ). At a larger scale, DSB formation occurs in the context of a specific chromosome architecture that consists of chromatin loops anchored to a proteinaceous axis (Zickler and Kleckner 1999 ). (springer.com)
  • DSB sites are preferentially located within chromatin loops, while several proteins that are required for DSB formation (Rec114, Mei4, and Mer2) localize on the chromosome axis (Blat et al. (springer.com)
Shear1
  • Novus Biologicals Visual Protocols: In phase 2 of this video you will learn how to effectively shear chromatin for ChIP using a sonicator, and how to analyze DNA fragment sizes with an agarose gel. (labtube.tv)
Modifiers1
  • Consistently, among 13 drugs targeting chromatin modifiers, EHMT2 inhibitors were highly effective in reducing ERMS cell viability. (elifesciences.org)
Experiments1
  • Abelson-transformed pre-B cell lines derived from these mice were used in chromatin immunoprecipitation experiments to identify regulatory sequences bound by E2A in the context of an early B lymphocyte environment. (duke.edu)
Preparation1
  • There are mainly two types of ChIP, primarily differing in the starting chromatin preparation. (wikipedia.org)
Regulation1
  • These observations provide new insight on antigenic variation by showing that chromatin remodeling is an intrinsic feature of BES regulation. (asm.org)
Promoter1
  • Active and silent rDNAs have distinct chromatin states at the promoter and within the transcribed region. (asm.org)
Structure1
  • Chromatin structure is dynamic, adopting a more condensed conformation at transcriptionally silent regions (closed chromatin) than at transcriptionally active regions (open chromatin) ( 6 ). (asm.org)
Transcription factor1
  • DNA meth-ylation represses transcription by interfering with transcription factor binding and indirectly by recruiting methyl-CpG-binding proteins and reducing chromatin remodeling activities ( 12 ). (spandidos-publications.com)
Binding1
  • To avoid dissociation of non-histone proteins from the chromatin binding site, it is necessary to incorporate a cross-linking step. (thermofisher.com)
Gene expression1
Widely1
Experimental technique1
Solution1
  • Although this method is very efficient in releasing chromatin into the solution, it has a number of limitations. (sigmaaldrich.com)
Profiles1
  • FiT-seq produces high-quality chromatin profiles particularly for methylated histone marks but is not optimized for H3K27ac profiling. (nih.gov)
Cross-linked1