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

Participants

Reaction Steps

Bovine serum albumin (100 mg) was dissolved in distilled water (2.0 ml) and the mixture was adjusted to a pH of 9 with 1N NaOH. Fluorescein isothiocyanate (FITC, 100 mg) in dimethylformamide (1.0 ml) was added by drops with stirring while maintaining the pH at 9 by addition of 1N NaOH. After all of the FITC was added, the mixture was stirred at room temperature for two hours. The solution was then dialysed in a cellulose dialyzing tube (MW 12,000-14,000) against 10 mM phosphate buffer (1.0 liter, pH 7) for two hours. The dialyzing tube was then dialysed against two changes of 0.9% aqueous sodium chloride (4.0 liters), for 19 hours each time, and then against three changes of 10% dimethylsulfoxide in 0.9% aqueous sodium chloride, for 24 hours each time. The final solution showed no sign of fluorescein in the dialysis solution. The resulting immunogen solution was then removed from the dialyzing tube and lyophilized to yield an orange solid. The immunogen was used according to standard procedures to produce a rabbit anti-fluorescein antibody capture reagent. The antibody was purified according to the procedure described in Example 1.c. above.

Provenance & Evidence