Affinity determination
Measure the dissociation constant of a ligand in minutes, with a tenth of a milligram of protein.
The question it answers
How strongly does this hit bind?
How it works
The photo-CIDNP signal of the ligand is recorded at several target concentrations. The decrease of the signal gives the dissociation constant.
[FIGURE: TITRATION CURVE]
Published figures
| Time per affinity | 5 to 15 min |
| Protein | 0.1 mg |
| Range | Micromolar to millimolar |
| Shown for | Peptides binding a PDZ domain, fragments binding PIN1 |
| Ligands that do not polarise | Measured by competition |
| Agreement | Signal reduction correlates with STD NMR |
What you need
- A light-coupled NMR spectrometer.
- A photosensitiser. Fluorescein is the standard.
- The ligand, or a photo-CIDNP-active competitor for it.
- The target protein. No isotope labelling.
Limits
The ligand must show photo-CIDNP, or compete with a ligand that does.