000174325 001__ 174325
000174325 005__ 20181203022620.0
000174325 022__ $$a1439-7633
000174325 0247_ $$2doi$$a10.1002/cbic.201100173
000174325 037__ $$aARTICLE
000174325 245__ $$aDevelopment of SNAP-tag fluorogenic probes for wash-free fluorescence imaging
000174325 269__ $$a2011
000174325 260__ $$c2011
000174325 336__ $$aJournal Articles
000174325 520__ $$aThe ability to specifically attach chemical probes to individual proteins represents a powerful approach to the study and manipulation of protein function in living cells. It provides a simple, robust and versatile approach to the imaging of fusion proteins in a wide range of experimental settings. However, a potential drawback of detection using chemical probes is the fluorescence background from unreacted or nonspecifically bound probes. In this report we present the design and application of novel fluorogenic probes for labeling SNAP-tag fusion proteins in living cells. SNAP-tag is an engineered variant of the human repair protein O(6)-alkylguanine-DNA alkyltransferase (hAGT) that covalently reacts with benzylguanine derivatives. Reporter groups attached to the benzyl moiety become covalently attached to the SNAP tag while the guanine acts as a leaving group. Incorporation of a quencher on the guanine group ensures that the benzylguanine probe becomes highly fluorescent only upon labeling of the SNAP-tag protein. We describe the use of intramolecularly quenched probes for wash-free labeling of cell surface-localized epidermal growth factor receptor (EGFR) fused to SNAP-tag and for direct quantification of SNAP-tagged β-tubulin in cell lysates. In addition, we have characterized a fast-labeling variant of SNAP-tag, termed SNAP(f), which displays up to a tenfold increase in its reactivity towards benzylguanine substrates. The presented data demonstrate that the combination of SNAP(f) and the fluorogenic substrates greatly reduces the background fluorescence for labeling and imaging applications. This approach enables highly sensitive spatiotemporal investigation of protein dynamics in living cells.
000174325 6531_ $$aDrug Design
000174325 700__ $$aSun, Xiaoli
000174325 700__ $$aZhang, Aihua
000174325 700__ $$aBaker, Brenda
000174325 700__ $$aSun, Luo
000174325 700__ $$aHoward, Angela
000174325 700__ $$aBuswell, John
000174325 700__ $$0244184$$aMaurel, Damien$$g182348
000174325 700__ $$0244188$$aMasharina, Anastasiya$$g188795
000174325 700__ $$0240057$$aJohnsson, Kai$$g123155
000174325 700__ $$aNoren, Christopher J.
000174325 700__ $$aXu, Ming-Qun
000174325 700__ $$aCorrêa, Ivan R.
000174325 773__ $$j12$$k14$$q2217-26$$tChembiochem : a European journal of chemical biology
000174325 909C0 $$0252027$$pLIP$$xU10102
000174325 909CO $$ooai:infoscience.tind.io:174325$$pSB$$particle
000174325 917Z8 $$x123155
000174325 937__ $$aEPFL-ARTICLE-174325
000174325 973__ $$aEPFL$$rNON-REVIEWED$$sPUBLISHED
000174325 980__ $$aARTICLE