In step one, the protein of interest (POI) is go-linked with the DNA website it binds to in an in vitro surroundings. Usually this is completed via a mild formaldehyde fixation this is reversible with warmth.
Then, the cells are lysed and the DNA is sheared by means of sonication or using micrococcal nuclease. This effects in double-stranded chunks of DNA fragments, commonly 1 kb or less in duration. Those that have been cross-linked to the POI shape a POI-DNA complicated.
In the following step, only these complexes are filtered out of the set of DNA fragments, the usage of an antibody particular to the POI. The antibodies may be connected to a strong floor, can also have a magnetic bead, or a few other physical assets that lets in separation of move-connected complexes and unbound fragments. This method is largely an immunoprecipitation (IP) of the protein. This can be carried out either by using using a tagged protein with an antibody against the tag (ex. FLAG, HA, c-myc) or with an antibody to the local protein.
The pass-linking of POI-DNA complexes is reversed (generally through heating) and the DNA strands are purified. For the rest of the workflow, the POI is now not essential.
After an amplification and denaturation step, the unmarried-stranded DNA fragments are categorized with a fluorescent tag inclusive of Cy5 or Alexa 647.
Finally, the fragments are poured over the surface of the DNA microarray, which is noticed with short, single-stranded sequences that cowl the genomic part of interest. Whenever a categorized fragment “unearths” a complementary fragment at the array, they will hybridize and form once more a double-stranded DNA fragment.