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 micro-arrays, 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