Estimating Cellular Abundances of Halo-tagged Proteins in Live Mammalian Cells by Flow Cytometry.

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 (e.g., 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.