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Site-specific labeling of proteins for single-molecule FRET measurements using genetically encoded ketone functionalities.
- Source :
-
Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2011; Vol. 751, pp. 3-15. - Publication Year :
- 2011
-
Abstract
- Studies of protein structure and function using single-molecule fluorescence resonance energy transfer (smFRET) benefit dramatically from the ability to site-specifically label proteins with small fluorescent dyes. Genetically encoding the unnatural amino acid (UAA) p-acetylphenylalanine is an efficient way to introduce commercially available fluorescent tags with high yield and specificity. This protocol describes the expression in Escherichia coli of proteins containing this UAA in response to the amber stop codon TAG. Proteins were purified with high yield and subsequently labeled with the hydroxylamine derivative of Alexa Fluor® 488 functioning as a fluorescent donor dye. The proteins were then labeled via maleimide coupling chemistry at a unique cysteine with the acceptor dye Alexa Fluor® 594 to yield a dual-labeled protein ready for subsequent smFRET observation.
- Subjects :
- Bacteriophage T4 enzymology
Binding Sites
Cysteine
Electrophoresis, Polyacrylamide Gel
Escherichia coli genetics
Hydroxylamine chemistry
Maleimides chemistry
Muramidase genetics
Muramidase isolation & purification
Oximes chemistry
Phenylalanine analogs & derivatives
Phenylalanine genetics
Phenylalanine metabolism
Recombinant Proteins chemistry
Recombinant Proteins genetics
Recombinant Proteins isolation & purification
Transformation, Genetic
Fluorescence Resonance Energy Transfer
Fluorescent Dyes chemistry
Ketones chemistry
Muramidase chemistry
Protein Engineering methods
Staining and Labeling methods
Subjects
Details
- Language :
- English
- ISSN :
- 1940-6029
- Volume :
- 751
- Database :
- MEDLINE
- Journal :
- Methods in molecular biology (Clifton, N.J.)
- Publication Type :
- Academic Journal
- Accession number :
- 21674321
- Full Text :
- https://doi.org/10.1007/978-1-61779-151-2_1