Outline of the ChIP Protocol

Stage 1 — chromatin immunoprecipitation

1. Crosslinking of proteins to genomic DNA
2. Cell lysis
3. DNA fragmentation using sonication
4. DNA:protein complexes immunoprecipitated using an antibody to the protein of interest
5. Protein removal

Stage 2 — identification of the DNA corresponding to the binding site of the protein of interest using OGT customised oligonucleotide microarray

1. Target preparation — DNA labelled using Fluor with Klenow
2. Hybridisation to an OGT microarray — a two colour array — experiments can be designed so that one colour generates a signal to the immunoprecipitated DNA fragments and the other generates a signal to control DNA. The control DNA can be represented by whole genome DNA or DNA immunoprecipitated from a cell containing a mutant protein
3. Scanning
4. Results — analysis and interpretation — those probes with a high test signal/control signal ratios indicate the positions where the protein of interest binds

If no antibody is available to the protein of interest an epitope tag can be considered. For example, a FLAG tag can be introduced into the gene resulting in a recombinant protein that can be immunoprecipitated using a FLAG antibody. In this type of experiment the control is represented by a non-FLAG protein.

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