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文献和实验contains the cytoplasmic fraction. The pellet is the nuclear fraction. 9. Resuspend nuclear pellet in 50 μl complete Cell Extraction Buffer for 30 minutes on ice with vortexing at 10 minute internals. 10. Centrifuge for 30 minutes at 14,000 x g
Preparation of nuclear extract and cytoplasmic extract
Solutions:Buffer A (Hypotonic Buffer): 1L10 mM HEPES pH 7.9 10 ml 1M HEPES,pH 7.91.5 mM MgCl2 1.5 ml 1M MgCl210 mM KCl3.33 ml 3M KCl0.5 mM DTT 500 µl 1M DTT0.2 mM PMSF 1 ml 0.2 M PMSFBuffer B (S100 extraction buffer): 500 ml0.3 M HEPES pH 7.9 150 ml
Detection of Cytoplasmic and Nuclear Functions of mTOR by Fractionation
rapamycin. These findings further demonstrate that the here presented and detailly described fractionation procedure is a valuable tool to study protein localization and cytoplasmic/nuclear protein shuttling in the context of expanding mTOR signalling.
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