In situ isolation of nuclei or nuclear proteins from adherent cells: a simple, effective method with less cytoplasmic contamination

[摘要] BackgroundIsolation of nuclei or nuclear proteins is a prerequisite for western blot, nuclear proteome profiling, and other evaluations of nuclear proteins. Here, we developed a simple method for in situ isolation of nuclei or nuclear proteins by in situ removing the extranuclear part of adherent cells via a classical nonionic detergent triton X-100.ResultsFirst, the feasibility of our method was confirmed by confocal microscopy, atomic force microscopy, scanning electron microscopy, dynamic light scattering, immunofluorescence imaging, and time-lapse dynamic observation. Next, the optimal concentration range (approximately 0.1–1% for ~ 10 min) of triton X-100 and the optimal treatment time (< 30 min) of 0.1–1% Triton X-100 for our method were determined via western blotting of eight extra-/intra-nuclear proteins. Subsequently, the effectiveness, sensitivity, and cytoplasmic contamination of our method were tested by investigating the levels of phosphorylated p65 (a NF-κB subunit) in the nuclei of endothelial or tumor cells treated with/without lipopolysaccharide (LPS) via western blotting and by comparing with a commercial nuclear protein extraction kit (a classical detergent-based method). The data show that compared with the commercial kit our method obtained a higher yield of total nuclear proteins, a higher pP65 level in both control and LPS groups, and much lower content of GAPDH (as a reference for cytoplasmic contamination) in nuclei.ConclusionsThe in situ isolation of nuclei or nuclear proteins from adherent cells in this study is a simple, effective method with less cytoplasmic contamination. This method/strategy has the potential of improving the quality of downstream evaluations including western blotting and proteomic profiling.

[发布日期] 2023-04-06

[关键词] Adherent cells;Human umbilical vein endothelial cells (HUVECs);Isolation of nuclei;Lipopolysaccharide (LPS);Nuclear proteins;Western blotting;Triton