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Nuclear protein extraction of human IMR-32 cells

WC Wen-Teng Chang
MH Ming-Yuan Hong
CC Chien-Liang Chen
CH Chi-Yuan Hwang
CT Cheng-Chieh Tsai
CC Chia-Chang Chuang
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IMR-32 cells were collected from the culture dish and centrifuged for 5 min at 500×g. The cell pellets were mixed with a 10-fold volume of buffer A (10 mM HEPES [pH 7.9], 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and one tablet of protease and phosphatase inhibitor [Thermo Scientific, USA]) and incubated on ice for 8 min followed by short centrifugation for 10 s at 12,000×g at 4 °C. The buffer A-treated cell pellets were then mixed with a two-fold volume of buffer C (20 mM HEPES [pH 7.9], 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, and one tablet of protease and phosphatase inhibitor) and incubated on ice for 16 min. After centrifugation at 12,000×g at 4 °C, the supernatant containing the nuclear protein of IMR-32 cells was collected and stored at − 80 °C.

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