Chromatin Immunoprecipitation Cloning Protocol

- from the Farnham lab. Click here for full protocol link.

Day 1

1.  Add formaldehyde directly to tissue culture media to a final concentration of 1%.  We generally use 1 x 107 cells per antibody per timepoint.  For cloning, multiple IPs are performed and pooled at later steps therefore we generally start with 1 x 108 cells.  Incubate adherent cells on a shaking platform and suspension cells on a stir plate for 10 minutes at room temperature. 

2.  Stop the crosslinking reaction by adding glycine to a final concentration of 0.125 M.  Continue to rock or spin at room temp for 5 minutes.

Yeast Chromatin Immunoprecipitation

Protocol from Hahn Lab. Click here for full protocol.

Chromatin IP Method

ChIP buffers

Notes on ChIP Method

Quantative PCR using ³²P dCTP

Chromatin Immunoprecipitation from Stem Cells

Click here to link to the video

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type .

0:11 Introduction11

0:27 Formaldehyde Crosslinking Cells27

2:22 Preparing Magnetic Beads142

4:13 Cell Sonication253

5:58 Chromatin Immunoprecipitation, Wash, and Elution358

8:53 Crosslink Reversal and DNA Purification533

9:58 Conclusion

The current state of chromatin immunoprecipitation.

Collas P. Mol Biotechnol. 2010 May;45(1):87-100.
Abstract
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.

ChIP Epigenetic Modifications

Using ChIP-based technologies to identify epigenetic modifications in disease-relevant cells.

Falk J. IDrugs. 2010 Mar;13(3):169-74.
Abstract
The effect of epigenetic modifications on the regulation of gene expression and the concomitant relationship to human diseases has become a key area of biological research in recent years. Studies have suggested that there is direct correlation between epigenetic modifications, such as histone methylation, histone acetylation and DNA methylation, and gene expression in disease-relevant cells, including cancer cells. The development of chromatin immunoprecipitation (ChIP)-based technologies, such as ChIP-chip and ChIP-Seq, has facilitated the high-throughput genome-wide mapping of epigenetic modifications that enable researchers to define the epigenome in disease-relevant cells and use comparative ChIP-based epigenetic mapping to correlate changes in epigenetic modifications with key physiological changes in disease-relevant tissues, including cancer cells, stem cells and T-cells. This feature review article provides insight into the nature of epigenetic modifications, the ChIP-based technologies that are available, and how such methods are being used to identify key epigenetic regulatory activities in medically relevant areas such as cancer and immunology.

Quantitative chromatin immunoprecipitation

Analysis of protein-DNA interactions during meiosis by quantitative chromatin immunoprecipitation (qChIP).

Mendoza MA, Panizza S, Klein F. Methods Mol Biol. 2009;557:267-83.

Abstract

During meiotic prophase a number of important events require recombination between maternal and paternal chromosomes, which is initiated through the introduction of DNA double-strand breaks (DSBs). The majority of DSBs, which mostly occur at so-called hotspots, have been located between cohesin binding sites. qChIP (chromatin immunoprecipitation quantified by real-time PCR) is a sensitive, accurate, and cost-efficient alternative to ChIP-on-Chip for the analysis of noncovalent protein-DNA interactions at defined binding sites in vivo. Here we use qChIP to study Mre11 binding to three chromosomal loci during meiosis. We show that Mre11 interacts with a known hotspot region (UpsilonCR048) in the R-band of chromosome III, but not with a cold region in the G-band (UpsilonCR011). Interestingly Mre11 binds to a cohesin binding site (UpsilonCR067), 20 kb distal to UpsilonCR048, with similar intensity as to the hotspot, despite the absence of DSBs in this region.