BACKGROUND: Isolation 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 .
RESULTS: First, 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.
CONCLUSIONS: The 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.