中山大学《细胞生物学》思考题 09.doc

Testing & Thinking of Cell Biology (缺 2、3) 1、 You are using a cameleon indicator to measure intracellular concentrations of Ca2+ . The indicator posed ofa central calmodulin domain, which converts from an extended form to a much pact form upon calcium binding , and two flanking fluorescent proteins , with CFP attached at one end and YFP attached at the other . You have expressed this indicator in cells and now wish to measure intracellular changes in Ca2+ concentration in response to the biological process you are studying . The instructions say to excite the cameleon at 440 nm and to measure emission at 535 nm . How do you suppose the cameleon indicator works? ◆知识点: ①报告蛋白② CFP ( Blue,Cyan) 、 GFP 和 YFP ③钙调蛋白④发射光谱( emission spectra )⑤激发光谱( excitation spectra ) ◆解题思维(分析能力): ① CFP 与 YFP 的作用② Ca2+ 与钙调蛋白的关系◆本题的来源: Miyawaki A, Llopis J, Heim R, McCafferty JM, Adams JA, Ikura M& Tsien RY (1997) Fluorescent indicators for Ca2+ based on green fluorescent protein and calmodulin. Nature 388, 882 – 887. 1、 You are studying the binding of proteins to the cytoplasmic face of cultured neuroblastoma (神经母细胞瘤) cells and have found a method that gives a good yield of inside-out vesicles from the plasma membrane. Unfortunately, your preparations are contaminated with variable amounts of right-side-out vesicles. Nothing you have tried avoids this variable contamination. A frie