Quantitation of fluorescence energy transfer between cell surface proteins via fluorescence donor photobleaching kinetics.
Quantitation of fluorescence energy transfer between cell surface proteins via fluorescence donor photobleaching kinetics.
复制标题
通过荧光供体光漂白动力学定量细胞表面蛋白之间的荧光能量转移。
DOI：
10.1016/s0006-3495(94)80549-1
复制
发表时间：
1994
影响因子：
3.4
通讯作者：
Barisas,BG
中科院分区：
文献类型：
--
作者：
Young,RM;Arnette,JK;Roess,DA;Barisas,BG
我们描述了光漂白荧光能量转移测量对单个活细胞的实际方面。T. M. Jovin及其同事（最近参见Kubitscheck et al. 1993. Biophys. J. 64：110）是基于当受体荧光团存在时供体荧光团的不可逆光漂白的降低速率。测量供体光漂白率的差异标记的细胞与供体（荧光素异硫氰酸酯共轭蛋白质），并与供体和受体（四甲基罗丹明共轭蛋白质）允许计算荧光能量转移效率。我们评估可能的方法，光漂白和能量转移的基本过程中的数据分析，并建议为此目的的最佳策略。单个小鼠B淋巴细胞结合不同比例的供体和受体结合物的四价伴刀豆球蛋白A（Con A）和二价琥珀酰Con A的干扰素能量转移通过这些方法进行了检查。对于Con A，观察到最大转移效率为0.49 +/- 0.02。在类似条件下，供体淬灭的流式细胞术测量得到0.54 +/-0.03的值。对于琥珀酰Con A，最大转移效率为0.36。为了提供具体的例子，在这种能量转移的测定所产生的数量，我们提出的例子，个别细胞的数据和动力学分析，人口速率常数分布，和误差估计所涉及的各种数量。
We describe practical aspects of photobleaching fluorescence energy transfer measurements on individual living cells. The method introduced by T. M. Jovin and co-workers (see, most recently, Kubitscheck et al. 1993. Biophys. J. 64:110) is based on the reduced rate of irreversible photobleaching of donor fluorophores when acceptor fluorophores are present. Measuring differences in donor photobleaching rates on cells labeled with donor only (fluorescein isothiocyanate-conjugated proteins) and with both donor and acceptor (tetramethylrhodamine-conjugated proteins) allows calculation of the fluorescence energy transfer efficiency. We assess possible methods of data analysis in light of the underlying processes of photobleaching and energy transfer and suggest optimum strategies for this purpose. Single murine B lymphocytes binding various ratios of donor and acceptor conjugates of tetravalent concanavalin A (Con A) and divalent succinyl Con A were examined for interlectin energy transfer by these methods. For Con A, a maximum transfer efficiency of 0.49 +/- 0.02 was observed. Under similar conditions flow cytometric measurements of donor quenching yielded a value of 0.54 +/- 0.03. For succinyl Con A, the maximum transfer efficiency was 0.36. To provide concrete examples of quantities arising in such energy transfer determinations, we present examples of individual cell data and kinetic analyses, population rate constant distributions, and error estimates for the various quantities involved.