Analysis of proteins stained by Alexa dyes

Shijun Huang, Houyi Wang, Christopher A. Carroll, Shirley J. Hayes, Susan T. Weintraub, Philip Serwer

Research output: Contribution to journalArticlepeer-review

16 Scopus citations

Abstract

Alexa dye staining of proteins is used for the fluorescence microscopy of single particles that are sometimes multimolecular protein complexes. To characterize the staining, post-staining determination must be made of which protein(s) in a complex have been Alexa-stained. The present communication describes the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for performing this determination. The Alexa-stained proteins are observed directly in gels by illumination with an ultraviolet transilluminator. The test multimolecular particle is bacteriophage T7. The protein capsid of T7 is a multimolecular complex that has both external and internal proteins. SDS-PAGE of Alexa-stained bacteriophage T7 produces fluorescent capsid proteins each of which usually comigrates with an unstained protein. However, one Alexa-induced modification of protein migration was observed by SDS-PAGE. Mass spectrometry shows that the protein with modified migration is the major protein of the outer shell of the T7 capsid. The procedures used are generally applicable. The distribution of Alexa staining among T7 capsid proteins depends on the size of the dye molecule used. The larger the dye molecule is, the greater the preference for external proteins.

Original languageEnglish (US)
Pages (from-to)779-784
Number of pages6
JournalELECTROPHORESIS
Volume25
Issue number6
DOIs
StatePublished - Mar 2004

Keywords

  • Agarose gel
  • Bacteriophage T7
  • Electrophoresis
  • Fluorescence detection
  • Mass spectrometry
  • Sodium dodecyl sulfate polyacrylamide gel

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry

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