Gautier, ArnaudJuillerat, AlexandreHeinis, ChristianReis Correa Jr., IvanKindermann, MaikBeaufils, FlorentJohnsson, Kai2008-06-102008-06-102008-06-10200810.1016/j.chembiol.2008.01.007https://infoscience.epfl.ch/handle/20.500.14299/26229WOS:000253671000009The visualization of complex cellular processes involving multiple proteins requires the use of spectroscopically distinguishable fluorescent reporters. We have previously introduced the SNAP-tag as a general tool for the specific labeling of SNAP-tag fusion proteins in living cells. The SNAP-tag is derived from the human DNA repair protein O6- alkylguanine-DNA alkyltransferase (AGT) and can be covalently labeled in living cells using O6-benzylguanine derivatives bearing a chemical probe. Here we report the generation of an AGT-based tag, named CLIP-tag, which reacts specifically with O2-benzylcytosine derivatives. Because SNAP-tag and CLIP-tag possess orthogonal substrate specificities, SNAP and CLIP fusion proteins can be labeled simultaneously and specifically with different molecular probes in living cells. We furthermore show simultaneous pulse-chase experiments to visualize different generations of two different proteins in one sample.text::journal::journal article::research article