The preparation and use of fluorescent-protein conjugates for microvascular research

Abstract

A procedure is described for making large quantities (100 ml) of fluorochrome-labeled albumin. Chromatographic techniques are described for the purification of commercial albumin (BSA) and the purification of albumin from serum. We report experimentally determined optimal conditions for the covalent attachment of fluorescent dyes (rhodamine isothiocyanate (RITC) and fluorescein isothiocyanate (FITC] to albumin. Subsequent removal of all unreacted fluorescent material (UFM) was achieved using charcoal adsorption. We observed no loss of protein following charcoal treatment. The final protein conjugate was analyzed by polyacrylamide gel electrophoresis, gel chromatography, and isoelectric focusing. The conjugates were determined to be free of UFM and homogeneous with respect to molecular weight. However, FITC conjugation lowered the average isoelectric point of albumin by 0.1 to 0.3 pH units. Illustrations of combining fluorescence microscopy with FITC-BSA and RITC-BSA to view microvascular phenomena in skeletal muscle and the heart are given. Knowledge of the biochemical characteristics of the fluorochrome employed is important for proper interpretation of experimental results using this technique.