Question

An experimental approach was taken to support the idea that both topoisomerase 2 and the other SAR binding protein bind the bases of the radial loops under more physiological conditions. Around this same time, a completely different experimental approach led to purification of a protein complex that included this second SAR-binding protein as one of its subunits.

i) What was this experimental approach?

ii)  Using this experimental approach the investigators did a specific experiment whose results suggested  that this protein complex not only localized at the bases of the radial loops but actually were functionally required for mitotic chromosome organization.

Describe that specific experiment:

Answer 1

The experimental approach used here is Chip (Chromatin immunoprecipitation) assay.

This reveals the interaction between the DNA and the protien in living cells. . The ChIP assay approach helps to evaluate DNA – protein interactions in living cells by treating the cells with formaldehyde or other crosslinking reagents to stabilize the downstream purification and detection interactions. Performing ChIP assays involves knowledge of the target protein and DNA sequence to be analyzed, as researchers must supply the DNA sequence of interest with an antibody against the protein of interest, and PCR primers. Through the antibody, the protein – DNA complex is selectively precipitated from the other genomic DNA fragments and protein – DNA complexes. The PCR primers allow precise amplification and sequence detection of target DNA.