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Fluorescence labeling of the C‐terminus of proteins with a puromycin analogue in cell‐free translation systems
Author(s) -
Nemoto Naoto,
Miyamoto-Sato Etsuko,
Yanagawa Hiroshi
Publication year - 1999
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(99)01474-x
Subject(s) - puromycin , fluorescence , translation (biology) , green fluorescent protein , chemistry , fluorescence anisotropy , biophysics , fluorescence correlation spectroscopy , biochemistry , fluorescence microscope , fluorescent protein , messenger rna , protein biosynthesis , microbiology and biotechnology , biology , gene , physics , quantum mechanics , membrane
We have developed a new method for the C‐terminus‐specific fluorescence labeling of proteins. This method is based on the experimental finding that a fluorescent puromycin analogue at lower concentrations bonds efficiently to the C‐terminus of mature proteins in cell‐free translation systems using mRNA without a stop codon. This labeling is performed under moderate conditions and its labeling efficiency is in the range of 50–95%. Here we demonstrate a protein‐protein interaction assay using fluorescence polarization measurement. This labeling method should also be useful for other rapid molecular interaction assays without purification of the labeled proteins, such as fluorescence correlation spectroscopy.