title: 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 250pmol in 20μl 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.
authors.affiliation: GE Healthcare Bio-Sciences, SE-751 84 Uppsala, Sweden. Electronic address: erik.bjerneld@ge.com.
authors.forename: Erik J
authors.initials: EJ
authors.lastname: Bjerneld
authors.affiliation: GE Healthcare Bio-Sciences, SE-751 84 Uppsala, Sweden.
authors.forename: Johan D
authors.initials: JD
authors.lastname: Johansson
authors.affiliation: GE Healthcare Bio-Sciences, SE-751 84 Uppsala, Sweden.
authors.forename: Ylva
authors.initials: Y
authors.lastname: Laurin
authors.affiliation: GE Healthcare Bio-Sciences, SE-751 84 Uppsala, Sweden.
authors.forename: Åsa
authors.initials: Å
authors.lastname: Hagner-McWhirter
authors.affiliation: GE Healthcare Bio-Sciences, SE-751 84 Uppsala, Sweden.
authors.forename: Ola
authors.initials: O
authors.lastname: Rönn
authors.affiliation: GE Healthcare Bio-Sciences, SE-751 84 Uppsala, Sweden.
authors.forename: Robert
authors.initials: R
authors.lastname: Karlsson
mesh_terms: D000818:Animals; D016466:CHO Cells; D002232:Carbocyanines / Q000737:chemistry*; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D007700:Kinetics; D057230:Limit of Detection; D011506:Proteins / Q000032:analysis* / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D002232:Carbocyanines; D011506:Proteins; C085321:cyanine dye 5
keywords: Cy5; Labeling; Protein; SDS–PAGE