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  • Chromatin immunoprecipitation for identifying transcription factor targets in keratinocytes. 19908003

    Protein-DNA interactions, such as those that are necessary for transcription, are critical in regulating cellular function and behavior. The identification of DNA sequences that interact with transcriptional regulatory proteins is an important step necessary to better understand the molecular mechanisms regulating gene expression. Chromatin immunoprecipitation (ChIP) is one such procedure that provides a snapshot of which transcription factors are occupying specific DNA sequences. This method allows one not only to determine whether a particular genomic region is occupied by transcription factors but also to identify specific regulatory sequences that potentially control expression of their target genes. Recently, ChIP has been combined with both microarray analysis and a new generation of sequencing allowing a true genome-wide examination of transcription factor binding. Identifying the exact DNA sequence that a transcriptional regulatory protein binds, the precise timing of this association, and what other factors are involved in these interactions are important steps that will shed light on the transcriptional control mechanisms that dictate the biology of all cells, including keratinocytes.
    Document Type:
    Reference
    Product Catalog Number:
    17-295
    Product Catalog Name:
    Chromatin Immunoprecipitation (ChIP) Assay Kit

  • Chromatin immunoprecipitation (ChIP) to assay dynamic histone modification in activated gene expression in human cells. 20729799

    In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue(1). They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers(2) and autoimmune diseases(3). It is well established that transcription is regulated by alterations to the chromatin template(4,5). These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation(6). Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.
    Document Type:
    Reference
    Product Catalog Number:
    16-157
    Product Catalog Name:
    Protein A Agarose/Salmon Sperm DNA, 2.5 mL

  • Chromatin immunoprecipitation assay. 15597545

    Association between proteins and DNA is crucial for many vital cellular functions such as gene transcription, DNA replication and recombination, repair, segregation, chromosomal stability, cell cycle progression, and epigenetic silencing. It is important to know the genomic targets of DNA-binding proteins and the mechanisms by which they control and guide gene regulation pathways and cellular proliferation. Chromatin immunoprecipitation (ChIP) is an important technique in the study of protein-gene interactions. Using ChIP, DNA-protein interactions are studied within the context of the cell. The basic steps in this technique are fixation, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. Although ChIP is a very versatile tool, the procedure requires the optimization of reaction conditions. Several modifications to the original ChIP technique have been published to improve the success and to enhance the utility of this procedure. This review addresses the critical parameters and the variants of ChiP as well as the different analytical tools that can be combined with ChIP to enable better understanding of DNA-protein interactions in vivo.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple

  • Chromatin immunoprecipitation assay for tissue-specific genes using early-stage mouse embryos. 21559006

    Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions. We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events. In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.5. Myogenin is a regulatory factor required for skeletal muscle differentiation. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.
    Document Type:
    Reference
    Product Catalog Number:
    12-370
    Product Catalog Name:
    Normal Rabbit IgG