The SNAP-tag labeling technology provides a simple, robust, and versatile approach to the imaging of fusion proteins for a
wide range of experimental applications. Owing to the specific and covalent nature of the labeling reaction, SNAP-tag is well suited for
the analysis and quantification of fused target protein using fluorescence microscopy techniques. In this report, we present our most recent
findings on the labeling of SNAP-tag fusion proteins both in vitro and in cell culture with SNAP-tag substrates derived from single
regioisomers of carboxyrhodamine dyes. Carboxyrhodamines are invaluable fluorescent dyes for biotechnology applications including
DNA sequencing, detection on microarrays, and fluorescence in situ hybridization. We found that SNAP-tag reacts preferentially with the
6-positional regioisomer of carboxyrhodamine fluorescent dyes, whereas the 5-regioisomer predominantly contributes to background
fluorescence. Our experimental study also indicates that benzylchloropyrimidine (CP) conjugates of 6-carboxyrhodamines exhibit a dramatic
increase in the signal-to-noise ratio of fluorescently labeled cellular proteins compared to the benzylguanine (BG) conjugates, presumably
due to higher cell permeability. These new SNAP-tag substrates based on pure 6-regioisomers can significantly improve fluorescence
labeling in live cells and should become powerful tools for bioimaging applications.
Keywords: Cell imaging, covalent labeling, fluorescent probes, protein modifications.
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