Although highly desirable, measuring absolute abundances of cellular proteins is a non-trivial undertaking. Proteins are macromolecules essential to cell, tissue, and organism structure, function and regulation. 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.Īssigning numbers to biological processes and to the molecules participating in them is of foremost importance to constrain models and attain a mechanistic and quantitative knowledge of biological phenomena ( 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. First, a cell line expressing the Halo-tagged protein of interest is grown and labeled side-by-side with our standard line.
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.
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). Accurate abundance measurements of cellular proteins are required to achieve a quantitative and predictive understanding of any biological process inside the cell.
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