Analysis of proteins stained by Alexa dyes.
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.
Department of Biochemistry, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA.
Shijun
S
Huang
Houyi
H
Wang
Christopher A
CA
Carroll
Shirley J
SJ
Hayes
Susan T
ST
Weintraub
Philip
P
Serwer
D000085:Acetates / Q000032:analysis / Q000737:chemistry; D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000737:chemistry; D036022:Capsid Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D002867:Chromones / Q000032:analysis / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry*; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009930:Organic Chemicals; D013194:Staining and Labeling; D013388:Succinimides / Q000737:chemistry / Q000378:metabolism
D000085:Acetates; C400304:Alexa 350; C492694:Alexa Fluor 488 carboxylic acid succinimidyl ester; C417664:Alexa594; D036022:Capsid Proteins; D002867:Chromones; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D013388:Succinimides