Sodium Carbonate → 2-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid

Participants

Reaction Steps

Two ml of Fab solution were brought to pH 9.0 by the addition of solid sodium carbonate (Na2CO3). A solution of 1.5 mg of fluorescein isothiocyanate (FITC) in 0.1 ml dry dimethylformamide (DMF) was slowly added (over 1/2 min.) with stirring at room temperature. After an additional 2 hrs., the solution was briefly clarified on a Brinkmann centrifuge (21/2 min.) and applied for separation into a column of Sephadex G-25 (0.9×15 cm) equilibrated with 0.05 M KPO4 buffer at pH 8.0. The yellow protein band (first band eluted) was collected. The final solution was 10.4 mg/ml in protein and degree of labeling 4.1 fluoresceins per Fab.

Provenance & Evidence