ChIP using plant samples
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实验概要
The immunoprecipitation (IP) of cross-linked chromatin with antibodies specific for certain histone modifications (chromatin immunoprecipitation, ChIP), followed by PCR to detect a potential enrichment or depletion of a DNA sequence of interest within IP fractions, constitutes an elegant and direct method to query specific chromatin states of individual genes.
实验原理
Eukaryotic chromatin is a complex of DNA and associated histone proteins that are involved in the higher order packaging of DNA into chromosomes. The chromatin state of a given DNA sequence influences transcriptional activity and replication timing and is regulated by potentially reversible covalent modifications of DNA and histones. Histone modifications at conserved lysine and arginine residues within the flexible N-terminal tails, such as phosphorylation, acetylation and methylation, specify a code that serves as an interaction platform with specific domains of chromatin-associated proteins. The immunoprecipitation (IP) of cross-linked chromatin with antibodies specific for certain histone modifications (chromatin immunoprecipitation, ChIP), followed by PCR to detect a potential enrichment or depletion of a DNA sequence of interest within IP fractions, constitutes an elegant and direct method to query specific chromatin states of individual genes and is already routinely used in many labs. In contrast to animal cells, however, plant cells have a rigid cell wall which poses limitations to the simple utilization of protocols established for animals. In this protocol, the method described in used to study histone modifications in the plant model organism Arabidopsis thaliana. This protocol is an adapted version of the original procedure published by Lawrence and co-workers.
This protocol describes how chromatin is prepared from Arabidopsis, which can subsequently be used for chromatin immunoprecipitation (ChIP). The exact chromatin concentration should be determined before starting the X-ChIP assay.
主要试剂
Extraction buffer 1
0.4 M Sucrose
10 mM Tris-HCl, pH 8.0
10 mM MgCl2
5 mM β-mercaptoethanol
Protease inhibitors
Extraction buffer 2
0.25 M Sucrose
10 mM Tris-HCl, pH 8.0
10 mM MgCl2
1% w/v Triton X-100
5 mM β-mercaptoethanol
Protease inhibitors
Extraction buffer 3
1.7 M Sucrose
10 mM Tris-HCl, pH 8.0
2 mM MgCl2
0.15% w/v Triton X-100
5 mM β-mercaptoethanol
Protease inhibitors
The immunoprecipitation (IP) of cross-linked chromatin with antibodies specific for certain histone modifications (chromatin immunoprecipitation, ChIP), followed by PCR to detect a potential enrichment or depletion of a DNA sequence of interest within IP fractions, constitutes an elegant and direct method to query specific chromatin states of individual genes.
实验原理
Eukaryotic chromatin is a complex of DNA and associated histone proteins that are involved in the higher order packaging of DNA into chromosomes. The chromatin state of a given DNA sequence influences transcriptional activity and replication timing and is regulated by potentially reversible covalent modifications of DNA and histones. Histone modifications at conserved lysine and arginine residues within the flexible N-terminal tails, such as phosphorylation, acetylation and methylation, specify a code that serves as an interaction platform with specific domains of chromatin-associated proteins. The immunoprecipitation (IP) of cross-linked chromatin with antibodies specific for certain histone modifications (chromatin immunoprecipitation, ChIP), followed by PCR to detect a potential enrichment or depletion of a DNA sequence of interest within IP fractions, constitutes an elegant and direct method to query specific chromatin states of individual genes and is already routinely used in many labs. In contrast to animal cells, however, plant cells have a rigid cell wall which poses limitations to the simple utilization of protocols established for animals. In this protocol, the method described in used to study histone modifications in the plant model organism Arabidopsis thaliana. This protocol is an adapted version of the original procedure published by Lawrence and co-workers.
This protocol describes how chromatin is prepared from Arabidopsis, which can subsequently be used for chromatin immunoprecipitation (ChIP). The exact chromatin concentration should be determined before starting the X-ChIP assay.
主要试剂
Extraction buffer 1
0.4 M Sucrose
10 mM Tris-HCl, pH 8.0
10 mM MgCl2
5 mM β-mercaptoethanol
Protease inhibitors
Extraction buffer 2
0.25 M Sucrose
10 mM Tris-HCl, pH 8.0
10 mM MgCl2
1% w/v Triton X-100
5 mM β-mercaptoethanol
Protease inhibitors
Extraction buffer 3
1.7 M Sucrose
10 mM Tris-HCl, pH 8.0
2 mM MgCl2
0.15% w/v Triton X-100
5 mM β-mercaptoethanol
Protease inhibitors
