A multi-parametric flow cytometric assay to analyze DNA–protein interactions

Arbab, Mandana; Mahony, Shaun; Cho, Hyunjii; Chick, Joel M.; Rolfe, Alex; Hoff, John Peter van; Morris, Viveca W.S.; Gygi, Steven P.; Maas, Richard L.; Gifford, David K.; Sherwood, Richard I.

doi:10.1093/nar/gks1034

Cited by 4 publications

(1 citation statement)

References 45 publications

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“…Analysis of the interactions is helpful to understand the regulatory mechanism of gene expressions, various cellular activities, and pathogenesis of diseases. Several methods have been used to study the interaction between DNA and protein, including, electrophoretic mobility shift assays (EMSA) , capillary electrophoresis , fluorescence resonance energy transfer , atomic force microscopy , electron microscopy , cyclic voltammetry , DNA immunoprecipitation , flow cytometry , localized Scattering Plasmon Resonance (LSPR) spectroscopy , DNA‐protein‐interaction (DPI)‐ELISA screen , single‐molecule Förster resonance energy transfer , a photoelectrochemistry .…”

Section: Introductionmentioning

confidence: 99%

Electrophoretic behavior of DNA‐methyl‐CpG‐binding domain protein complexes revealed by capillary electrophoreses laser‐induced fluorescence

et al. 2015

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The free solution electrophoretic behavior of DNA-protein complexes depends on their charge and mass in a certain experimental condition, which are two fundamental properties of DNA-protein complexes in free solution. Here, we used CE LIF to study the free solution behavior of DNA-methyl-CpG-binding domain protein (MBD2b) complexes through exploring the relationship between the mobilities, charge, and mass of DNA-protein complexes. This method is based on the effective separation of free DNA and DNA-protein complexes because of their different electrophoretic mobility in a certain electric field. In order to avoid protein adsorption, a polyacrylamide-coated capillary was used. Based on the evaluation of the electrophoretic behavior of formed DNA-MBD2b complexes, we found that the values of (μ0 /μ)-1 were directly proportional to the charge-to-mass ratios of formed complexes, where the μ0 and μ are the mobility of free DNA probe and DNA-protein complex, respectively. The models were further validated by the complex mobilities of protein with various lengths of DNA probes. The deviation of experimental and calculated charge-to-mass ratios of formed complexes from the theoretical data was less than 10%, suggesting that our models are useful to analyze the DNA-binding properties of the purified MBD2b protein and help to analyze other DNA-protein complexes. Additionally, this study enhances the understanding of the influence of the charge-to-mass ratios of formed DNA-protein complexes on their separation and electrophoretic behaviors.

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Section: Introductionmentioning

confidence: 99%

Electrophoretic behavior of DNA‐methyl‐CpG‐binding domain protein complexes revealed by capillary electrophoreses laser‐induced fluorescence

et al. 2015

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A DNA-Centric Protein Interaction Map of Ultraconserved Elements Reveals Contribution of Transcription Factor Binding Hubs to Conservation

et al. 2013

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Ultraconserved elements (UCEs) have been the subject of great interest because of their extreme sequence identity and their seemingly cryptic and largely uncharacterized functions. Although in vivo studies of UCE sequences have demonstrated regulatory activity, protein interactors at UCEs have not been systematically identified. Here, we combined high-throughput affinity purification, high-resolution mass spectrometry, and SILAC quantification to map intrinsic protein interactions for 193 UCE sequences. The interactome contains over 400 proteins, including transcription factors with known developmental roles. We demonstrate based on our data that UCEs consist of strongly conserved overlapping binding sites. We also generated a fine-resolution interactome of a UCE, confirming the hub-like nature of the element. The intrinsic interactions mapped here are reflected in open chromatin, as indicated by comparison with existing ChIP data. Our study argues for a strong contribution of protein-DNA interactions to UCE conservation and provides a basis for further functional characterization of UCEs.

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Future Applications of Flow Cytometry and Related Techniques

Béné¹,

Lacombe²

Multiparameter Flow Cytometry in the Diagnosis of Hematologic Malignancies

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A multi-parametric flow cytometric assay to analyze DNA–protein interactions

Cited by 4 publications

References 45 publications

Electrophoretic behavior of DNA‐methyl‐CpG‐binding domain protein complexes revealed by capillary electrophoreses laser‐induced fluorescence

Electrophoretic behavior of DNA‐methyl‐CpG‐binding domain protein complexes revealed by capillary electrophoreses laser‐induced fluorescence

A DNA-Centric Protein Interaction Map of Ultraconserved Elements Reveals Contribution of Transcription Factor Binding Hubs to Conservation

Future Applications of Flow Cytometry and Related Techniques

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