No-wash protein labeling with designed fluorogenic probes and application to real-time pulse-chase analysis.
Small 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.
Division of Advanced Science and Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Shin
S
Mizukami
Shuji
S
Watanabe
Yuri
Y
Akimoto
Kazuya
K
Kikuchi
D002462:Cell Membrane / Q000737:chemistry; D002511:Cephalosporins / Q000737:chemistry; D019793:Fluorescein / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D011726:Pyridinium Compounds / Q000737:chemistry; D001618:beta-Lactamases / Q000737:chemistry
D002511:Cephalosporins; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D011506:Proteins; D011726:Pyridinium Compounds; D001618:beta-Lactamases; D019793:Fluorescein