title: Substrates for improved live-cell fluorescence labeling of SNAP-tag.
abstract: 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.
authors: New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA. correa@neb.com
authors: Ivan R
authors: IR
authors: Corrêa
authors: Brenda
authors: B
authors: Baker
authors: Aihua
authors: A
authors: Zhang
authors: Luo
authors: L
authors: Sun
authors: Christopher R
authors: CR
authors: Provost
authors: Gražvydas
authors: G
authors: Lukinavičius
authors: Luc
authors: L
authors: Reymond
authors: Kai
authors: K
authors: Johnsson
authors: Ming-Qun
authors: MQ
authors: Xu
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D057054:Molecular Imaging / Q000379:methods*; D015394:Molecular Structure; D010539:Permeability; D012235:Rhodamines / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D005456:Fluorescent Dyes; D012235:Rhodamines