The biotin/avidin system is a fantastic affinity-based purification method, well known in the biochemistry field. Compared to other affinity-based method, it has two major advantages:
- Biotin/avidin binding is the strongest non-covalent interaction known in nature (Kd=10-15M)
- There are few naturally biotinylated proteins in mammalian cells.
During biotinylation, BirA ligase from E. coli adds a biotin moiety to the lysine residue of the 23 AA tag added either at the N-terminal or C-terminal end of the modified target protein. A one-step purification step is sufficient for protein complex identification by MS. Alternatively biotinylated proteinscan be cross linked to DNA for ChIPseq analysis.
We established this system in mouse embryonal stem cells initially to investigate the mechanism of Sall4 action. In the meantime we have succesfully tagged numerous additional proteins and generated "biotinylation knock-in mice" (adapted from).
This powerful tool helps us understand more about key transcription regulators in vivo by comparing their function in different organs i.e. brain, pancreas and kidney.