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丁香实验推荐阅读
Sedimentation and Immunoprecipitation Assays for Analyzing Complexes that Repress Transcription

Co-repressor proteins function as platforms for the assembly of multi-subunit complexes that mediate transcriptional repression. Common components of such complexes are histone deacetylases, which catalyze the removal of acetyl groups from the tails of histones within nucle ...

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Isolation of Nuclei for Use in Genome-Wide DNase Hypersensitivity Assays to Probe Chromatin Structure

DNase hypersensitivity (DHS) analysis coupled with high-throughput DNA sequencing (DNase-seq) has emerged as a powerful tool to analyze chromatin accessibility and identify regulatory sequences in genomic DNA on a global scale. In this method, intact nuclei are isolated from fresh t ...

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Histone Deacetylase Inhibitor Valproic Acid as a Small Molecule Inducer to Direct the Differentiation of Pluripotent Stem Cells

Pluripotent stem cells can be directed into myogenic differentiation by small molecular inducers, which preferentially activate muscle-specific transcription networks. Here we describe how to efficiently direct the differentiation of pluripotent P19 cells into skelet ...

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Use of Histone Deacetylase Inhibitors to Examine the Roles of Bromodomain and Histone Acetylation in p300-Dependent Gene Expression

The bromodomain is an evolutionarily conserved motif harbored by many transcription regulators and nearly all nuclear histone acetyltransferases including the transcriptional coactivator p300. The function of p300 is required for the expression of an array of genes, in part thro ...

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Analysis of p300 Occupancy at the Early Stage of Stem Cell Differentiation by Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) is an invaluable method to study the specific interaction of regulatory proteins with genomic DNA. Since its first development, it has been modified extensively to make it applicable to many different cell types and experimental systems. The cro ...

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Heavy Methyl-SILAC Labeling Coupled with Liquid Chromatography and High-Resolution Mass Spectrometry to Study the Dynamics of Site-Specific Histone Me

Histone lysine and arginine methylation involved in gene activation and silencing is dynamically regulated. However, partly limited to the research technologies previously available, the dynamics of global histone methylation on a site-specific basis have not been fully pursu ...

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Simple and Efficient Identification of Chromatin Modifying Complexes and Characterization of Complex Composition

Affinity purification and mass spectrometry analysis have been used to identify and characterize protein complexes. Wdr82-associated chromatin modifying complexes were purified by single-step FLAG affinity purification from human cells induced to express FLAG-tagged W ...

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Immunoaffinity Purification of Protein Complexes from Mammalian Cells

In this chapter, we describe a purification scheme designed to isolate multisubunit protein complexes gently and quickly from crude extracts of mammalian cells using immunoaffinity purification of epitope tagged proteins and the multisubunit complexes with which they associ ...

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Peptide Microarrays for Profiling of Serine/Threonine Kinase Activity of Recombinant Kinases and Lysates of Cells and Tissue Samples

Peptide microarray technology can be used to identify substrates for recombinant kinases, to measure kinase activity and changes thereof in cell lysates and lysates from fresh frozen (tumor) tissue. The effect of kinase inhibitors on the kinase activities in relevant tissues can be inve ...

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Preparation of Cell Lines for Single-Cell Analysis of Transcriptional Activation Dynamics

Imaging molecularly defined regions of chromatin in single living cells during transcriptional activation has the potential to provide new insight into gene regulatory mechanisms. Here, we describe a method for isolating cell lines with multi-copy arrays of reporter transgenes, w ...

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Gene Regulation

This review concisely highlights the complexity of regulatory events. It provides examples of how interconnectivity of regulatory hubs could maintain transcriptional synergy and orchestrate the proper spatial and temporal patterns of gene expression.

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Nuclear Recruitment Assay as a Tool to Validate Transcription Factor Interactions in Mammalian Cells

Identification and verification of novel transcription factor interactions is an inherent step in the discovery of molecular mechanisms driving gene transcription and regulation. Co-immunoprecipitation and GST-pull down are often key techniques in the verification proc ...

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Fluorescence Cross-correlation Spectroscopy (FCCS) to Observe Dimerization of Transcription Factors in Living Cells

Fluorescence cross-correlation spectroscopy (FCCS) is an established spectroscopic method to observe the interaction between the different fluorescent molecules. Using FCCS, researchers can assess the interaction of target molecules in the aqueous condition, and can app ...

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Promoter Independent Abortive Transcription Assays Unravel Functional Interactions Between TFIIB and RNA Polymerase

TFIIB-like general transcription factors are required for transcription initiation by all eukaryotic and archaeal RNA polymerases (RNAPs). TFIIB facilitates both recruitment and post-recruitment steps of initiation; in particular, TFIIB stimulates abortive initiati ...

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Using FRET to Monitor Protein-Induced DNA Bending: The TBP-TATA Complex as a Model System

Proteins that bind to DNA can elicit changes in DNA conformation, such as bending and looping, which are important signals for later events such as transcription. TATA-binding protein (TBP) is one example of a protein that elicits a conformational change in DNA; TBP binds and sharply bends its reco ...

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Chromatin Assembly and In Vitro Transcription Analyses for Evaluation of Individual Protein Activities in Multicomponent Transcriptional Complexes

Eukaryotic DNA and core histones form the fundamental repeating units of chromatin. Condensed c�hromatin, which has higher-order structures, prevents transcriptional complexes from accessing their target genes. Epigenetic regulation, including structural changes of c ...

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Quantitative NanoProteomics Approach for Protein Complex (QNanoPX) Using Gold Nanoparticle-Based DNA Probe

Affinity purification by pulldown methods using target-bound gel beads provides a powerful approach for purifying endogenous protein complexes. Such methods can be improved by using nanoparticle-based probe, coupled with immunoblot analysis or quantitative proteomics me ...

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Combination of Native and Denaturing PAGE for the Detection of Protein Binding Regions in Long Fragments of Genomic DNA

In traditional electrophoresis mobility shift assay (EMSA) a single 32P-labeled double-stranded DNA oligonucleotide or a restriction fragment bound to a protein is separated from the unbound DNA by polyacrylamide gel electrophoresis (PAGE) under nondenaturing conditions. ...

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Electrophoretic Mobility-Shift and Super-Shift Assays for Studies and Characterization of ProteinDNA Complexes

Gene expression is in part regulated by transcription factors that bind specific sequence motifs in genomic DNA. Transcription factors cooperate with the basal machinery to upregulate or downregulate transcription. Experimental data have revealed the importance of interact ...

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Identifying Specific ProteinDNA Interactions Using SILAC-Based Quantitative Proteomics

A comprehensive identification of protein–DNA interactions that drive processes such as transcription and replication, both in prokaryotic and eukaryotic organisms, remains a major technical challenge. In this chapter, we present a SILAC-based DNA affinity purification met ...

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