The conjugation of immunoglobulins with tetramethylrhodamine isothiocyanate. A comparison between the amorphous and the crystalline fluorochrome.

L Amante, A Ancona, L Forni

Index: J. Immunol. Methods 1 , 289, (1972)

Full Text: HTML

Abstract

Methods are described for the conjugation of small amounts of immunoglobulins with amorphous or crystalline tetramethylrhodamine isothiocyanate (TRITC); the conjugation is performed at protein concentrations not exceeding 4 mg/ml. Conjugation with the amorphous fluorochrome results in a high recovery (up to 87%) of conjugated proteins suitable for immunofluorescence work, whereas lower recovery (50–56%) is obtained with the crystalline fluorochrome. The loss of proteins in the latter case is due to denaturation caused by the acetone used to dissolve crystalline TRITC which is insoluble in aqueous solutions. Conjugates made with amorphous TRITC have to be purified by gel-filtration followed by DEAE-cellulose chromatography, while only gel-filtration is needed to purify conjugates made with crystalline TRITC. For the latter, it is possible to calculate the number of fluorochrome groups bound per protein molecule.