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Isolation of nuclear and cytoplasmic extract

CL Chang Ho Lee
KK Kyung Woon Kim
DL Da-hye Lee
SL So Min Lee
SK So Young Kim
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To estimate the expression levels of nuclear and cytosolic RAGE, nuclear and cytoplasmic fractions were extracted using an NE-PER Nuclear Cytoplasmic Extraction Reagent kit (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. Briefly, the temporal cortex tissue was washed twice with cold PBS and centrifuged at 500g for 5 min. The pellet was resuspended in 400L of cytoplasmic extraction reagent I by homogenizing. The suspension was then incubated on ice for 10 min followed by the addition of 22L of cytoplasmic extraction reagent II. The mixture was vortexed for 5 s, incubated on ice for 1 min and centrifuged for 5 min at 16000g. The supernatant (cytoplasmic extract) was transferred to a microcentrifuge tube. The insoluble pellet was resuspended in 200L of nuclear extraction reagent by vortexing for 15 s, incubated on ice for 10 min, and centrifuged for 10 min at 16000g. The resulting supernatant constituted the nuclear extract.

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