Cattoglio Claudia, Darzacq Xavier, Tjian Robert, Hansen Anders S
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.
Li Ka Shing Center for Biomedical and Health Sciences, Berkeley, CA, USA.
Bio Protoc. 2020 Feb 20;10(4):e3527. doi: 10.21769/BioProtoc.3527.
Accurate abundance measurements of cellular proteins are required to achieve a quantitative and predictive understanding of any biological process inside the cell. Existing methods to determine absolute protein abundances are labor-intensive and/or require sophisticated experimental and computational infrastructure (, fluorescence correlation spectroscopy (FCS)-calibrated imaging and quantitative mass spectrometry). Here we detail a straightforward flow cytometry-based method to measure the absolute abundance of any Halo-tagged protein in live cells that uses a standard mammalian cell line with a known number of Halo-CTCF proteins recently characterized in our lab. The protocol only comprises a few steps. First, a cell line expressing the Halo-tagged protein of interest is grown and labeled side-by-side with our standard line. Then, average fluorescence intensities are measured by conventional flow cytometry analysis and finally a simple calculation is applied to estimate the absolute number of the Halo-tagged protein of interest per cell. Once the protein of interest has been endogenously tagged with HaloTag, which we routinely achieve by Cas9-mediated genome editing, the presented protocol is fast, convenient, reproducible, cost-effective and readily accessible.
要对细胞内的任何生物过程实现定量且可预测的理解，就需要准确测量细胞蛋白质的丰度。现有的确定绝对蛋白质丰度的方法既耗费人力，又需要复杂的实验和计算基础设施（例如，荧光相关光谱法（FCS）校准成像和定量质谱分析）。在此，我们详细介绍一种基于流式细胞术的直接方法，用于测量活细胞中任何带有 Halo 标签的蛋白质的绝对丰度，该方法使用我们实验室最近鉴定出的具有已知 Halo-CTCF 蛋白数量的标准哺乳动物细胞系。该方案仅包含几个步骤。首先，将表达感兴趣的 Halo 标签蛋白的细胞系与我们的标准细胞系并排培养并标记。然后，通过常规流式细胞术分析测量平均荧光强度，最后进行简单计算以估计每个细胞中感兴趣的 Halo 标签蛋白的绝对数量。一旦通过 Cas9 介导的基因组编辑将感兴趣的蛋白质内源性标记上 HaloTag（我们通常采用这种方法），所介绍的方案就快速、便捷、可重复、经济高效且易于操作。
