Labeling of Antibodies with Cy3-, Cy3.5-, Cy5-, and Cy5.5-monofunctional Dyes at Defined Dye/Protein Ratios

Abstract

In a recent study [Gruber et al. (2000) Bioconjugate Chem. 11, 696 – 704] the fluorescence signal per labeled antibody has been related to the number of bound Cy dye per protein molecule. Unexpectedly, a strong anomalous fluorescence enhancement was observed when Cy3 or Cy3.5 were covalently bound to protein, whereas fluorescence loss occurred when Cy5 or Cy5.5 were attached to protein. Moreover, up to eight Cy3 or Cy3.5 molecules could be bound per antibody molecule before the cumulative fluorescence output reached a maximum, while labeling of antibodies with more than three Cy5 molecules was counterproductive. These findings are of particular relevance to the use of Cy dye-labeled proteins in single molecule fluorescence microscopy. The manuscript for the already published article originally contained detailed information on how to adjust the average numbers of dyes per antibody. These protocols, however, became obsolete when the commercial supplier changed the quantity and quality (much higher succinimdyl ester content) of the Cy dye portions “to label 1 mg protein”. The present study describes the adjustment of the desired Cy dye/IgG ratios by variation of protein concentration, dye concentration, and pH in the labeling reaction, based upon the new, purer Cy dye reagents. In combination with the already published study, these data allow to aim at optimal fluorescence of labeled antibody by chosing proper labeling conditions. The complete poster can be viewed at: http://www.bphys.uni-linz.ac.at/bioph/download/label.pdf.