通过定量荧光显微镜术测定绝对蛋白质数量。
Determining absolute protein numbers by quantitative fluorescence microscopy.
作者信息
Verdaasdonk Jolien Suzanne, Lawrimore Josh, Bloom Kerry
机构信息
Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
出版信息
Methods Cell Biol. 2014;123:347-65. doi: 10.1016/B978-0-12-420138-5.00019-7.
Biological questions are increasingly being addressed using a wide range of quantitative analytical tools to examine protein complex composition. Knowledge of the absolute number of proteins present provides insights into organization, function, and maintenance and is used in mathematical modeling of complex cellular dynamics. In this chapter, we outline and describe three microscopy-based methods for determining absolute protein numbers--fluorescence correlation spectroscopy, stepwise photobleaching, and ratiometric comparison of fluorescence intensity to known standards. In addition, we discuss the various fluorescently labeled proteins that have been used as standards for both stepwise photobleaching and ratiometric comparison analysis. A detailed procedure for determining absolute protein number by ratiometric comparison is outlined in the second half of this chapter. Counting proteins by quantitative microscopy is a relatively simple yet very powerful analytical tool that will increase our understanding of protein complex composition.
生物学问题越来越多地通过使用广泛的定量分析工具来研究蛋白质复合物的组成。了解存在的蛋白质的绝对数量有助于深入了解其组织、功能和维持情况，并用于复杂细胞动力学的数学建模。在本章中，我们概述并描述了三种基于显微镜的确定蛋白质绝对数量的方法——荧光相关光谱法、逐步光漂白法以及荧光强度与已知标准的比例比较法。此外，我们还讨论了用作逐步光漂白和比例比较分析标准的各种荧光标记蛋白质。本章后半部分概述了通过比例比较确定蛋白质绝对数量的详细程序。通过定量显微镜对蛋白质进行计数是一种相对简单但非常强大的分析工具，它将增进我们对蛋白质复合物组成的理解。
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