Pre-labeling of diverse protein samples with a fixed amount of Cy5 for sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.

Abstract A pre-labeling protocol based on Cy5 N -hydroxysuccinimide (NHS) ester labeling of proteins has been developed for one-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis. We show that a fixed amount of sulfonated Cy5 can be used in the labeling reaction to label proteins over a broad concentration range—more than three orders of magnitude. The optimal amount of Cy5 was found to be 50 to 250 pmol in 20 μl using a Tris–HCl labeling buffer at pH 8.7. Labeling protein samples with a fixed amount of dye in this range balances the requirements of sub-nanogram detection sensitivity and low dye-to-protein (D/P) ratios for SDS–PAGE. Simulations of the labeling reaction reproduced experimental observations of both labeling kinetics and D/P ratios. Two-dimensional electrophoresis was used to examine the labeling of proteins in a cell lysate using both sulfonated and non-sulfonated Cy5. For both types of Cy5, we observed efficient labeling across a broad range of molecular weights and isoelectric points.