Chromatin Immunoprecipitation (CHIP)
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These are numerous protein that
function by interacting directly with DNA, such chromatin protein or the factor
that perform replication, repair and transcription
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It is critical to map the dynamics of
protein-DNA interaction in living cell in order to fully understand the complex
function.
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The powerful technique of chromatin
immunoprecipitation (CHIP) was developed to capture such interaction.
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CHIP allows to reaches to detect the
presence of any protein of interest at a specific DNA sequence in vivo.
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It allows the detection of specific
protein-DNA interaction in vivo.
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CHIP on CHIP or CHIP sequence allows mapping
of all the protein-binding sites for a given protein across the entire genome.
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This method depends on the use of an
antibody to detect the protein interest.
Protocol: -
1.
Chromatin immunoprecipitation (CHIP)
away of discovery whether a given protein is bound to given chromatin, the
DNA-protein complex that is the natural state of DNA in living cell.
2.
One start with chromatin isolated
from cell and adds formaldehyde to from covalent bond between DNA and any
protein bond to it.
3.
The one shears to chromatin by
sonication to produce short dsDNA fragment cross-linked to protein.
4.
Next, one makes cell extracts and
immunoprecipitation the protein DNA complex with antibodies directed against a
protein of interest.
5.
This precipitation that specific
protein and the DNA to which it binds.
6.
To see it that DNA contains the gene
of interest, one performs immunoprecipitate with primer designed to amplify
that gene.
7.
If the gene is present, a DNA
fragment of predicated size will and be detectable as a bond after gel
electrophoresis.
Advantages: -
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In combination with microarray that
represent the genome (CHIP on CHIP), the assay become much more powerful,
identifying the binding sequence and its location in the genome by
hybridization to a particular micro-array feature.
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