Characterization of a biological detector cell for quantitation of inositol 1,4,5-trisphosphate.
Characterization of a biological detector cell for quantitation of inositol 1,4,5-trisphosphate.
复制标题
用于定量肌醇 1,4,5-三磷酸的生物检测器单元的表征。
DOI：
10.1006/abio.1999.4390
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
Allbritton,NL
中科院分区：
文献类型：
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作者：
Luzzi,V;Murtazina,D;Allbritton,NL
测量单细胞中的肌醇1，4，5-三磷酸（IP 3）的先前策略要么是定性的，要么具有有限的空间分辨率。毛细管电泳结合生物检测细胞已被用来定量IP 3在非洲爪蟾卵母细胞的小区域。为了提高该方法的检测限，我们阐明了影响IP 3检测池耦合到毛细管电泳的灵敏度和可靠性的实验参数。影响检测器单元的变量是跨检测器单元的电压降的幅度、该电压降的持续时间、毛细管中的流体流动的方向、检测器单元周围的游离Ca 2+的浓度以及检测器单元的透化期间蛋白酶抑制剂的存在。对于由毛细管直径施加的样品体积，检测器单元主要充当IP 3质量检测器而不是浓度检测器。表征的实验变量影响的灵敏度和可靠性，这种检测器单元有可能提高其他分析物的测量进行配对毛细管电泳与生物检测器单元。
Prior strategies to measure inositol 1,4,5-trisphosphate (IP3) in single cells either have been qualitative or have had a limited spatial resolution. Capillary electrophoresis combined with a biological detector cell has been used to quantitate IP3in small regions of a Xenopus oocyte. To improve the detection limits of this method, we elucidated the experimental parameters which influenced the sensitivity and reliability of the IP3–detector cell coupled to capillary electrophoresis. The variables which influenced the detector cell were the magnitude of the voltage drop across the detector cell, the duration of this voltage drop, the direction of fluid flow in the capillary, the concentration of free Ca2+around the detector cell, and the presence of protease inhibitors during permeabilization of the detector cell. For the sample volumes imposed by the capillary diameter, the detector cell acted primarily as an IP3mass detector rather than a concentration detector. Characterization of the experimental variables influencing the sensitivity and reliability of this detector cell has the potential to enhance other analyte measurements performed by mating capillary electrophoresis with a biological detector cell.