Xenopus oocyte plasma membrane sheets for FRET analysis.
Xenopus oocyte plasma membrane sheets for FRET analysis.
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用于 FRET 分析的爪蟾卵母细胞质膜片。
DOI：
10.1152/ajpcell.00435.2006
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发表时间：
2007
期刊：
影响因子：
--
通讯作者：
John,Scott
中科院分区：
文献类型：
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作者：
Ottolia,Michela;Philipson,KennethD;John,Scott
从异种卵母细胞质膜片已被分离用于荧光共振能量转移（FRET）测量。该系统具有以下优点：1）从大表面积进行荧光记录以最大化信噪比，2）减少来自保留在细胞内隔室中的蛋白质的背景荧光，以及3）进入质膜的细胞质表面以快速更换溶液。为了证明这种方法的实用性，我们已经研究了以前发表的FRET为基础的Ca 2+传感器，即Cameleon-PM。该构建体靶向质膜，并且在各种Ca 2+添加到膜的细胞质面时，显示比率FRET变化。根据记录的比率变化，确定表观Ca 2+亲和力为1.65 μM。因此，制备异种卵母细胞质膜片和FRET测量证明了上述所有三个优点。
Plasma membrane sheets fromXenopusoocytes have been isolated for use in fluorescence resonance energy transfer (FRET) measurements. This system has the following advantages:1) fluorescent recordings from a large surface area to maximize the signal-to-noise ratio,2) reduction in background fluorescence from proteins retained in intracellular compartments, and3) access to the cytoplasmic surface of the plasma membrane for rapid solution changes. To demonstrate the utility of this approach, we have examined a previously published FRET-based Ca2+sensor, namely, the Cameleon-PM. This construct targets to the plasma membrane and, upon various Ca2+additions to the cytoplasmic face of the membrane, shows ratiometric FRET changes. From the ratiometric changes recorded, an apparent Ca2+affinity of 1.65 μM was determined. Thus preparation ofXenopusoocyte plasma membrane sheets and FRET measurements demonstrates all three of the advantages outlined above.