Root D D
Department of Biological Sciences, University of North Texas, P.O. Box 5218, Denton, TX 76203-5218, USA.
Proc Natl Acad Sci U S A. 1997 May 27;94(11):5685-90. doi: 10.1073/pnas.94.11.5685.
A novel method was developed to detect molecular associations of dystrophin with actin in cryostat muscle tissue sections by combining resonance energy transfer technology with immunohistochemical techniques. This method takes advantage of the long phosphorescent lifetime of terbium chelates, a property that enables the accurate determination of energy transfer in biological tissues by lifetime measurements of sensitized emission. After a brief excitation pulse, terbium chelates emit for milliseconds after the intrinsically high autofluorescence of biological specimens has decayed to negligible levels. Rat skeletal muscle tissue sections were labeled with both anti-dystrophin monoclonal antibody conjugated to a terbium-based resonance energy transfer donor and anti-actin tetramethylrhodamine phalloidin as an acceptor. Resonance energy transfer between the two probes indicated that the distance separating the probes is within 10 nm (about the size of an IgG2b antibody molecule). The fraction of antibodies that participated in resonance energy transfer was estimated to be 80-90% because of the close agreement between the quenching of donor phosphorescence and the efficiency of resonance energy transfer revealed by lifetime measurements of sensitized emission by tetramethyl-rhodamine phalloidin. Sensitized emission was detectable only when both anti-dystrophin antibody and tetramethyl-rhodamine phalloidin were present. These results indicate that actin and dystrophin are closely associated within the cell. This method is potentially applicable to the investigation of many types of intracellular associations.
通过将共振能量转移技术与免疫组织化学技术相结合，开发出一种新方法来检测恒冷箱肌肉组织切片中肌营养不良蛋白与肌动蛋白的分子关联。该方法利用了铽螯合物较长的磷光寿命，这一特性能够通过敏化发射的寿命测量来准确测定生物组织中的能量转移。在短暂的激发脉冲后，铽螯合物在生物标本本身较高的自发荧光衰减到可忽略不计的水平后仍会发射数毫秒。用与基于铽的共振能量转移供体偶联的抗肌营养不良蛋白单克隆抗体和作为受体的抗肌动蛋白四甲基罗丹明鬼笔环肽对大鼠骨骼肌组织切片进行标记。两种探针之间的共振能量转移表明，探针之间的距离在10纳米以内（约为IgG2b抗体分子的大小）。由于供体磷光猝灭与四甲基罗丹明鬼笔环肽敏化发射寿命测量所揭示的共振能量转移效率之间密切一致，估计参与共振能量转移的抗体比例为80 - 90%。只有当抗肌营养不良蛋白抗体和四甲基罗丹明鬼笔环肽都存在时，才能检测到敏化发射。这些结果表明，肌动蛋白和肌营养不良蛋白在细胞内紧密关联。该方法有可能适用于多种类型细胞内关联的研究。
