ja208290f_si_001.pdf (893.96 kB)
No-Wash Protein Labeling with Designed Fluorogenic Probes and Application to Real-Time Pulse-Chase Analysis
journal contribution
posted on 2012-01-25, 00:00 authored by Shin Mizukami, Shuji Watanabe, Yuri Akimoto, Kazuya KikuchiSmall molecule labeling techniques for cellular proteins
under
physiological conditions are very promising for revealing new biological
functions. We developed a no-wash fluorogenic labeling system by exploiting
fluorescence resonance energy transfer (FRET)-based fluorescein-cephalosporin-azopyridinium
probes and a mutant β-lactamase tag. Fast quencher elimination,
hydrophilicity, and high resistance against autodegradation were achieved
by rational refinement of the structure. By applying the probe to
real-time pulse-chase analysis, the trafficking of epidermal growth
factor receptors between cell surface and intracellular region was
imaged. In addition, membrane-permeable derivatization of the probe
enabled no-wash fluorogenic labeling of intracellular proteins.