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Preparation of purified nuclei from mammalian cells (isotonic + NP-40 method)
Posted by: Vania, Northwestern University, Chicago, IL
This protocol is based on the procedure described by Wagschal A et al (doi:10.1101/pdb.prot4767 Cold Spring Harb Protoc 2007) and allows the preparation of highly purified nuclei. Since NP-40 is used instead of dounce homogenization for lysing cells, the resulting nuclei will be cleaner and homogeneus. However, some nuclear proteins may be extracted during the cell lysis step.
Materials
10x Nuclei Isolation (10x NI): 150 mM Tris-HCl pH 7.4, 150 mM NaCl, 600 mM KCl, 50 mM MgCl2, 1 mM EGTA