Bamezai Anil, Kennedy Colleen
Department of Biology, Villanova University, Villanova, PA, USA.
Methods Mol Biol. 2008;477:137-47. doi: 10.1007/978-1-60327-517-0_12.
Cholesterol-rich microdomains present on the plasma membrane appear to play an important role in spatio-temporal regulation of cell signaling and cell adhesion processes. Compositional heterogeneity of these microdomains and their coalescence during cell-cell interactions may provide one mechanism for triggering and/or regulating signaling cascades from the plasma membrane to the cell interior. Biochemical analyses of distinct lipid microdomain subpopulations and single-rafts obtained from unstimulated and ligand-stimulated cells are critical for deciphering functional role of lipid rafts. We have designed a cell-free assay that captures detergent-resistant lipid rafts with an antibody against a raft-resident molecule and detects the presence of another lipid raft molecule. Moreover, this cell-free assay provides a simple and quick way to examine the simultaneous presence of two proteins in the lipid rafts, and has the potential to estimate trafficking of molecules in and out of the lipid microdomains during cell signaling on a single lipid raft-basis.
存在于质膜上富含胆固醇的微结构域似乎在细胞信号传导和细胞黏附过程的时空调节中发挥重要作用。这些微结构域的组成异质性及其在细胞间相互作用过程中的聚结可能提供一种机制，用于触发和/或调节从质膜到细胞内部的信号级联反应。对从未受刺激和配体刺激的细胞中获得的不同脂质微结构域亚群和单个脂筏进行生化分析，对于解读脂筏的功能作用至关重要。我们设计了一种无细胞测定法，该方法用针对脂筏驻留分子的抗体捕获抗去污剂脂质筏，并检测另一种脂质筏分子的存在。此外，这种无细胞测定法提供了一种简单快速的方法来检查脂质筏中两种蛋白质的同时存在，并且有可能在单个脂质筏基础上估计细胞信号传导过程中分子进出脂质微结构域的运输情况。
