As such, its expression level is stable and reflects endogenous CTCF. This is a U2OS human clonal cell line (clone C32) with the endogenous CTCF locus homozygously engineered via Cas9-mediated genome editing to express a Halo-CTCF protein. In fact, we first generated what is here used as a standard cell line with the intent of studying CTCF nuclear dynamics by single-molecule super-resolution microscopy ( ), and since it is currently the preferred choice for live-cell single-molecule imaging (
) because of its popularity and versatility, with applications in a broad range of experimental systems ( We focused on the HaloTag protein-fusion platform (
The method has several advantages over existing ones: it employs a human U2OS-derived cell line standard that is easy to culture it only requires a flow cytometer, an instrument available in most institutions it has single-cell resolution it can be reproducibly repeated multiple times with little effort and cost and, it involves very basic data analysis. ), with the ultimate goal of deciphering the spatiotemporal regulation of transcription and its interplay with 3D genome organization. This was part of a larger effort to quantify nuclear transcription factors and architectural 3D-genome regulators like CTCF and cohesin ( We have recently developed a new flow cytometry-based absolute quantification method that can be easily applied to any Halo-tagged protein in the cell. ) are less complex, but still rely on accurate cell counting and/or depend on the availability of a pure recombinant protein to use as a calibrator. Electrophoresis-based methods ( e.g., traditional SDS-PAGE or more advanced capillary systems followed by staining/blotting ( However, it again requires advanced microscopy equipment and computational infrastructure. Quantitative FCS-calibrated imaging is a live cell method that can be streamlined to achieve high-throughput abundance measurement during dynamic cellular processes, while retaining single-cell resolution, and can be selective for different sub-cellular compartments ( This is certainly the case for quantitative mass spectrometry, a powerful yet complex technology that allows high-throughput measurements of un-modified proteins ( The few methods currently available are time-consuming, costly and technically challenging, and for the most part rely on complicated equipment and analytical tools not readily accessible to most laboratories. 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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