Fluorescein labeling of Fab' while preserving single thiol.

We determined the conditions for labeling rabbit Fab' with fluorescein isothiocyanate to provide maximal fluorescence intensity compatible with the preservation of integrity of its single thiol residue. We found that we could reproducibly substitute Fab' with two fluorescein moieties in 30 min at room temperature at pH 8.9. We achieved a high level of fluorescence intensity while we preserved the integrity of more than half of the existing thiols.