Fluorometric identification and microdetermination of proteins labeled with 4-(3′-phenyl-2′-pyrazolin-1′-yl)benzenesulfonyl chloride in polyacrylamide gel matrices

Abstract

Protein-dodecyl sulfate complexes, labeled at room temperature for 15 min under slightly basic conditions with 4-(3′-phenyl-2′-pyrazolin-1′-yl)benzenesulfonyl chloride, exhibit a fluorescence emission band centered at 420 nm. Their fluorescence intensity is independent of pH in the range 2–8. In polyacrylamide gel electrophoresis, their relative mobilities are not different from their unlabeled counterparts. The limit of detection for protein in solution is 2 μg/ml. Following electrophoresis of 10-μl sample volumes in 5-mm-diameter gel cylinders, and without extrusion from the glass tubes, the typical detection limit of the labeled protein-dodecyl sulfate complexes is 250 ng. The rate of increase of the fluorescence signal with increasing amount of labeled protein-dodecyl sulfate complex in the sample volume varies from one protein to another. The linear range for these growth curves is also different. These differences are related to the lysine content of each protein.