Chip‐on‐beads: Flow‐cytometric evaluation of chromatin immunoprecipitation

Székvölgyi, Lóránt; Bálint, Bálint László; Imre, László; Goda, Katalin; Szabó, Miklós; Nagy, László; Szabó, Gábor

doi:10.1002/cyto.a.20325

Cited by 11 publications

(7 citation statements)

References 18 publications

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“…Data from this report show that FCIP can be also used to analyze TCR‐CD3 interactions. The use of flow cytometry has previously been used to investigate BCR‐Abl fusion proteins in clinical leukaemia samples (21) and to perform chromatin immunoprecipitation (chIP) analysis (22). In this report, it is initially shown that single‐color flow cytometry can be used to analyze interactions between CD3ζ‐scFv fusion molecules and the TCR.…”

Section: Discussionmentioning

confidence: 99%

Development of a flow cytometric co‐immunoprecipitation technique for the study of multiple protein—protein interactions and its application to T‐cell receptor analysis

et al. 2009

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Co-immunoprecipitation is the classical approach for investigating protein-protein interactions. Analysis is generally conducted using the Western blot approach. We set out to investigate whether flow cytometry was a feasible alternative to Western blotting. Using the TCR-CD3 complex as a model for intermolecular interactions in the MA5.8 cell line, FLAG-tagged CD3f-scFv fusion proteins could be captured on anti-FLAG coupled beads and associated TCRb molecules could be detected by flow cytometry. This association was abrogated by mutations to the CD3f transmembrane domain. Using multicolor flow cytometry, TCRb, CD3e, and the scFv region of the CD3f fusion molecule could all be detected from a single sample. This multicolor analysis was then applied to demonstrate the importance of correct lysis conditions for extraction of the TCR complex. In summary, this flow cytometric immunoprecipitation technique is a feasible alternative to classical co-immunoprecipitation analysis technique and offers many potential advantages including rapid analysis with increased target sensitivity, reduced technical demands, amenable to multiple protein analysis from a single sample, and provides a framework that may facilitate the development of high throughput analytical assays investigating protein-protein interactions. ' 2009 International Society for Advancement of Cytometry Key termsT-cell receptor; CD3f; co-immunoprecipitation; protein-protein interactions THE co-immunoprecipitation/Western blotting (co-ip/WB) approach has been the gold-standard technique for the investigation of suspected protein-protein interactions for many years. However, the co-ip/WB approach has limitations. For example, heavy and light chains from the capture antibodies can obscure identification of immuno-reactive bands on immuno-blots of the same molecular weight as the protein of interest. Furthermore, SDS-PAGE/WB can be a time-consuming process, particularly if multiple protein interactions are being investigated. To circumvent some of the issues surrounding standard co-ip/WB, we investigated the use of flow cytometry to analyze immunoprecipitates from a well-documented protein complex. The advantage of this approach being that the high throughput approach of flow cytometry would expedite the identification process and, with the large number of fluorochrome conjugated antibodies now available to the researcher, would permit analysis of multiple protein-protein interactions. Bead-based assays have steadily become adapted for flow cytometric purposes and can now be used to analyze kinase inhibitors (1), to quantify antibody binding sites (2), to detect low concentrations of DNA (3), and to measure cytokine secretion (4). Here, we show that a bead-based flow cytometric immunoprecipitation assay (FCIP) can be used to successfully analyze interactions between CD3f and the T-cell receptor (TCR) complex. Not only is

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Section: Discussionmentioning

confidence: 99%

Development of a flow cytometric co‐immunoprecipitation technique for the study of multiple protein—protein interactions and its application to T‐cell receptor analysis

et al. 2009

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“…In the recent special issue in Cytometry Part A (Vol. 69A, Issue 5, 2006), a number of reports investigating bead‐based analysis for a range of applications were published including protein interactions (28, 29), high‐resolution single particle analysis (30), GST‐pull down assays (29), low concentration hybridization assays (31), and chromatin immunoprecipitation (32). This highlights the intense interest in using bead‐based flow cytometric assays across a range of disciplines but also highlights the need for standardized procedures for extracting quantitative data.…”

Section: Discussionmentioning

confidence: 99%

Quantitative data analysis methods for bead‐based DNA hybridization assays using generic flow cytometry platforms

Corrie¹,

Lawrie²,

Battersby³

et al. 2008

Cytometry Pt A

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Bead-based assays are in demand for rapid genomic and proteomic assays for both research and clinical purposes. Standard quantitative procedures addressing raw data quality and analysis are required to ensure the data are consistent and reproducible across laboratories independent of flow platform. Quantitative procedures have been introduced spanning raw histogram analysis through to absolute target quantitation. These included models developed to estimate the absolute number of sample molecules bound per bead (Langmuir isotherm), relative quantitative comparisons (two-sided ttests), and statistical analyses investigating the quality of raw fluorescence data. The absolute target quantitation method revealed a concentration range (below probe saturation) of Cy5-labeled synthetic cytokeratin 19 (K19) RNA of c.a. 1 3 10 4 to 500 3 10 4 molecules/bead, with a binding constant of c.a. 1.6 nM. Raw hybridization frequency histograms were observed to be highly reproducible across 10 triplex assay replicates and only three assay replicates were required to distinguish overlapping peaks representing small sequence mismatches. This study provides a quantitative scheme for determining the absolute target concentration in nucleic acid hybridization reactions and the equilibrium binding constants for individual probe/target pairs. It is envisaged that such studies will form the basis of standard analytical procedures for bead-based cytometry assays to ensure reproducibility in inter-and intra-platform comparisons of data between laboratories. '

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“…Importantly, labeling must occur in the linear phase of the PCR to ensure reliable quantification. The similarity of the data obtained by qPCR and flow cytometry has been shown for the enrichment of H4 and H3 epitopes on a specific locus in Jurkat cells [68].…”

Section: Flow Cytometry Analysis Of Chip Dna: Chip-on-beadsmentioning

confidence: 93%

“…The ChIP-on-beads assay has been proposed to be useful for quantitative assessments of ChIP products in a highthroughput manner [68]. However, the complexity of the procedure makes it at present difficult to foresee the advantage of ChIP-on-beads over ChIP-qPCR or ChIP-onchip approaches, especially as long as a qPCR analysis of ChIP products is necessary for evaluation of the linear phase of the PCR-mediated labeling step.…”

Section: Flow Cytometry Analysis Of Chip Dna: Chip-on-beadsmentioning

confidence: 99%

“…A recent protocol, however, calls for the capture of conventional PCR products on microbeads and flow cytometry analysis [68]. A standard ChIP is performed, and the ChIP DNA is used as template for end-point PCR in which primers are tagged in their 5 0 end with Fam (forward primer) and biotin (reverse primer).…”

Section: Flow Cytometry Analysis Of Chip Dna: Chip-on-beadsmentioning

confidence: 99%

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The Current State of Chromatin Immunoprecipitation

2010

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The biological significance of interactions of nuclear proteins with DNA in the context of gene expression, cell differentiation, or disease has immensely been enhanced by the advent of chromatin immunoprecipitation (ChIP). ChIP is a technique whereby a protein of interest is selectively immunoprecipitated from a chromatin preparation to determine the DNA sequences associated with it. ChIP has been widely used to map the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin modifying enzymes on the genome or on a given locus. In spite of its power, ChIP has for a long time remained a cumbersome procedure requiring large numbers of cells. These limitations have sparked the development of modifications to shorten the procedure, simplify sample handling and make ChIP amenable to small numbers of cells. In addition, the combination of ChIP with DNA microarray and high-throughput sequencing technologies has in recent years enabled the profiling of histone modification, histone variants, and transcription factor occupancy on a genome-wide scale. This review highlights the variations on the theme of the ChIP assay, the various detection methods applied downstream of ChIP, and examples of their application.

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Chip‐on‐beads: Flow‐cytometric evaluation of chromatin immunoprecipitation

Cited by 11 publications

References 18 publications

Development of a flow cytometric co‐immunoprecipitation technique for the study of multiple protein—protein interactions and its application to T‐cell receptor analysis

Development of a flow cytometric co‐immunoprecipitation technique for the study of multiple protein—protein interactions and its application to T‐cell receptor analysis

Quantitative data analysis methods for bead‐based DNA hybridization assays using generic flow cytometry platforms

The Current State of Chromatin Immunoprecipitation

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