CelLytic™ NuCLEAR™ Extraction Kit

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	The nucleus and cytoplasm of MHV-68-infected BHK-21 cells were isolated using a CelLytic NuCLEAR extraction kit (product code NXTRACT; Sigma-Aldrich) according to the instructions in the manual accompanying the kit, with some modifications.

Protocol tips
The nucleus and cytoplasm of MHV-68-infected BHK-21 cells were isolated using a CelLytic NuCLEAR extraction kit (product code NXTRACT; Sigma-Aldrich) according to the instructions in the manual accompanying the kit, with some modifications.

Publication protocol

The nucleus and cytoplasm of MHV-68-infected BHK-21 cells were isolated using a CelLytic NuCLEAR extraction kit (product code NXTRACT; Sigma-Aldrich) according to the instructions in the manual accompanying the kit, with some modifications. Briefly, eight 15-cm plates of confluent BHK-21 cells were infected at an MOI of 5. At 12 h postinfection, the cells were digested with trypsin, collected by centrifugation at 1,000 × g for 5 min, resuspended in 6 ml of lysis buffer (containing dithiothreitol [DTT] and protease inhibitors; product code L9036; Sigma-Aldrich), and incubated on ice for 5 min. Then, 10% Igepal CA-630 solution was added to the swollen cells to a final concentration of 0.1%, and the mixture was vortexed vigorously for 10 s. The cytoplasmic fraction was separated from the nuclei by centrifugation at 1,000 × g for 10 min. The nuclei were washed three times and resuspended in 0.5 ml of extraction buffer (product code E2525; Sigma-Aldrich) containing DTT and protease inhibitors. Capsids were released from the purified nuclei by freezing (80°C) and then thawing (37°C) three times. Insoluble materials from the nuclear and cytoplasmic fractions were cleared by centrifugation at 8,000 × g for 30 min. The capsids remaining in the soluble supernatant of the nuclear and cytoplasmic fractions were pelleted through a 1.7-ml 30% (wt/vol) sucrose cushion in TNE buffer (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM EDTA) by centrifugation at 83,500 × g for 1 h in a P40T rotor (Hitachi). The pellets were then resuspended in 500 μl of TNE buffer, sonicated for 2 min at moderate power, and layered onto a discontinuous sucrose density gradient consisting of 20 to 45% (wt/vol) sucrose in TNE buffer. The gradients were then centrifuged at 74,000 × g for 1 h in a P40T rotor (Hitachi). All centrifugation steps were carried out at 4°C. Fractions of 850 μl each were collected from the top of the gradient. A total of 14 fractions, named fractions 1 to 14, were obtained from the top to the bottom. Trichloroacetic acid (TCA) was added to a final concentration of 13%, and the samples were incubated overnight at 4°C. The precipitated proteins were collected by centrifugation at 18,000 × g for 30 min, washed with 100% ethanol, and resuspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer for Western blotting.

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