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Analysis of proteins stained by Alexa dyes
Author(s) -
Huang Shijun,
Wang Houyi,
Carroll Christopher A.,
Hayes Shirley J.,
Weintraub Susan T.,
Serwer Philip
Publication year - 2004
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200305723
Subject(s) - alexa fluor , staining , capsid , sodium dodecyl sulfate , fluorescence , polyacrylamide gel electrophoresis , chemistry , gel electrophoresis , biophysics , fluorescence microscope , chromatography , microbiology and biotechnology , biochemistry , biology , enzyme , genetics , physics , quantum mechanics , gene
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.