The smFRET and FCS capabilities of the EI-FLEX can both be employed to investigate the binding and dissociation of biomolecular complexes, the formation of aggregates and to perform studies of the competitive binding of small molecules - such as for small molecule drug screens.
The use of confocal single-molecule methods to study protein binding and dissociation offers several benefits:
- No surface immobilisation of the protein is required
- No flow-cell or complex experimental set-up is required
- Experiments can be performed under biologically relevant conditions
- Work directly with cell lysates without extensive purification
- Work at very low concentration (picomolar)
- Accurately measure very low dissociation constants (kDs)
Further reading
https://pubs.rsc.org/en/content/articlehtml/2020/nr/d0nr01060j
Fig 1. Overlays of the FCS correlation curves for free dye, a dye labelled antibody and a complex i.e. bound protein or vesicle containing the labelled antibody. As size increases so does diffusion time, which is the basis of the FCS technique.