期刊
NATURE METHODS
卷 9, 期 10, 页码 987-+出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/NMETH.2174
关键词
-
资金
- US National Institutes of Health (NIH) [GM077248]
- American Cancer Society [RSG0823401GMC]
- Netherlands Organization for Scientific Research (NWO) [Vici 680-47-607]
- NIH [GM62267]
- National Institute of General Medical Sciences Molecular Biophysics Training Grant [T32 GM008313]
Single-molecule fluorescence imaging is often incompatible with physiological protein concentrations, as fluorescence background overwhelms an individual molecule's signal. We solve this problem with a new imaging approach called PhADE (PhotoActivation, Diffusion and Excitation). A protein of interest is fused to a photoactivatable protein (mKikGR) and introduced to its surface-immobilized substrate. After photoactivation of mKikGR near the surface, rapid diffusion of the unbound mKikGR fusion out of the detection volume eliminates background fluorescence, whereupon the bound molecules are imaged. We labeled the eukaryotic DND replication protein flap endonuclease 1 with mKikGR and added it to replication-competent Xenopus laevis egg extracts. PhADE imaging of high concentrations of the fusion construct revealed its dynamics and micrometer-scale movements on individual, replicating DNA molecules. Because PhADE imaging is in principle compatible with any photoactivatable fluorophore, it should have broad applicability in revealing single-molecule dynamics and stoichiometry of macromolecular protein complexes at previously inaccessible fluorophore concentrations.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据