High-throughput fluorescence labelling of full-length cDNA products based on a reconstituted translation system.
Although recent advances in fluorescence-based technologies, such as protein microarrays, have made it possible to analyse more than 10,000 proteins at once, there is a bottleneck in the step of preparation of large numbers of fluorescently labelled proteins for the comprehensive analysis of protein-protein interactions. Here we describe two independent methods for high-throughput fluorescence-labelling of full-length cDNA products at their C-termini using a reconstituted translation system containing fluorescent puromycin. For the first method, release factor-free systems were used. For the second method, stop codons were excluded from cDNAs by using a common mismatch primer in mutagenic PCR. These methods yielded large numbers of labelled proteins from cDNA sets of various organisms, such as mouse, yeast and Escherichia coli.
Kawahashi
Yuko
Y
Department of Biosciences and Informatics, Keio University, Yokohama, Japan.
Doi
Nobuhide
N
Oishi
Yo
Y
Tsuda
Chizuru
C
Takashima
Hideaki
H
Baba
Tomoya
T
Mori
Hirotada
H
Ito
Takashi
T
Yanagawa
Hiroshi
H
D002232:Carbocyanines / Q000737:chemistry; D002474:Cell-Free System / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D009097:Multienzyme Complexes / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D014176:Protein Biosynthesis; D040901:Proteomics / Q000379:methods; D011691:Puromycin / Q000031:analogs & derivatives*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism
D002232:Carbocyanines; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D009097:Multienzyme Complexes; D029701:Saccharomyces cerevisiae Proteins; C089187:cyanine dye 3; D011691:Puromycin; C073439:PUP2 protein, S cerevisiae; D046988:Proteasome Endopeptidase Complex