Reaction #214356

Reaction equation

NaOH

fluorescein isothiocyanate

aluminum

HCl

→

fluorescein

Conditions

Workup

1. 1
   Other180mgs of BSA (2.6×10-6Moles) (Pentex, crystallized)
2. 2
   workup.DISSOLUTIONdissolved in 6mls H2O containing 180mgs K2CO3
3. 3
   Washeluted with 0.05M PO4, pH 8.0 at 5.4 mls/hr, 0.9 ml/fraction
4. 4
   OtherHapten number was calculated from the UV absorption of the conjugate at 493 Mμ

Procedure

Into a scintillation vial was placed 180mgs of BSA (2.6×10-6Moles) (Pentex, crystallized) dissolved in 6mls H2O containing 180mgs K2CO3. To this was added 18.3mgs fluorescein isothiocyanate (FITC) (4.85×10-5Moles) and the mixture was stoppered, covered with aluminum foil then placed in the center hole of a vortex mixer and shaken gently overnight at room temperature. The following morning the reaction mixture was acidified to pH 4 with 1N HCl (heavy precipitate), then made basic to pH 8 with 0.1N NaOH, applied to a 2.5×30cm column of Sephadex G-10 and eluted with 0.05M PO4, pH 8.0 at 5.4 mls/hr, 0.9 ml/fraction. Fractions 43-72 were pooled. Hapten number was calculated from the UV absorption of the conjugate at 493 Mμ and the extinction coefficient for protein-bound fluorescein of 7.2×104. Hapten number was thus calculated to be 14.5.

Source