NE-PER™ Nuclear and Cytoplasmic Extraction Reagents

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- The cells were resuspended in RIPA buffer and lysed by sonication at 40% power with four 10-second pulses and a 20 second rest on ice.	- For nuclear and cytoplasmic protein isolation, the Thermo Fisher NE-PER kit was used according to the manufacturer’s instructions with the exception of isolating nuclear proteins with sonication at 40% power with two 10-second pulses and 20 second rest on ice.

Upstream tips
- The cells were resuspended in RIPA buffer and lysed by sonication at 40% power with four 10-second pulses and a 20 second rest on ice.
Protocol tips
- For nuclear and cytoplasmic protein isolation, the Thermo Fisher NE-PER kit was used according to the manufacturer’s instructions with the exception of isolating nuclear proteins with sonication at 40% power with two 10-second pulses and 20 second rest on ice.

Publication protocol

The cells were resuspended in RIPA buffer and lysed by sonication at 40% power with four 10-second pulses and a 20 second rest on ice. For nuclear and cytoplasmic protein isolation, the Thermo Fisher NE-PER kit was used according to the manufacturer’s instructions with the exception of isolating nuclear proteins with sonication at 40% power with two 10-second pulses and 20 second rest on ice. Lysates were run on a 7.5% SDS-PAGE gels. Following semi-dry transfer or wet transfer (for visualization of NIK) to immobilon-P, samples were analyzed by Western blotting for p100/p52 (Cell Signaling), p105/p50 (Millipore), actin (Santa Cruz Biotechnology), laminB1 (Cell Signaling), tubulin (Cell Signaling), and NIK (Santa Cruz Biotechnology). Densitometric quantification of the bands was performed using Image Lab software (Bio-Rad).

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