Fluorescence labeling of the C-terminus of proteins with a puromycin analogue in cell-free translation systems.
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.
Nemoto
N
N
Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo, Japan.
Miyamoto-Sato
E
E
Yanagawa
H
H
D002474:Cell-Free System; D005452:Fluoresceins / Q000737:chemistry*; D005453:Fluorescence; D005454:Fluorescence Polarization / Q000379:methods*; D011485:Protein Binding; D014176:Protein Biosynthesis; D011691:Puromycin / Q000031:analogs & derivatives* / Q000737:chemistry*
D005452:Fluoresceins; C402634:fluorpuro; D011691:Puromycin