Techniques Flashcards
1
Q
How does CUT&RUN work
A
A specific antibody to the protein of interest and pAGMNase is used to isolate protein-DNA complexes
1. Cells are first harvested and bound to concavalin beads to minimize cell loss in subsequent washes
2. Cells are permeability’s with digitonin to allow for primary antibody to enter and bind to protein of interest
3. The pAG domain of PAG-MNases binds to the heavy chain of th eprimary antibody- so the enzyme is on the area of chromatin of interest
4. With the addition of Ca2+, the enzyme is activated- DNA digestion results
5. The cleaved chromatin complex of interest then leaves the genetic material and can be collected
6. Purified DNA can be used to construct a seuqencing library for NGS or whole genome screening
