CelLytic™ NuCLEAR™ Extraction Kit

Protocol tips

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	For Western blot analysis, nuclear and cytoplasmic fractions were generated using the CelLytic NuCLEAR Extraction kit (Cat#NXTRACT Sigma-Aldrich,). 48 hours after transfection, cells were washed with PBS, collected, and resuspended in 0.5 ml of hypotonic lysis buffer and incubated on ice for 15 minutes, 30 μl of 10% IGEPAL was added to the cell suspension. Cells were then vortexed and spun at 10,000 xg for 30 seconds at 4°C.

Protocol tips

For Western blot analysis, nuclear and cytoplasmic fractions were generated using the CelLytic NuCLEAR Extraction kit (Cat#NXTRACT Sigma-Aldrich,). 48 hours after transfection, cells were washed with PBS, collected, and resuspended in 0.5 ml of hypotonic lysis buffer and incubated on ice for 15 minutes, 30 μl of 10% IGEPAL was added to the cell suspension. Cells were then vortexed and spun at 10,000 xg for 30 seconds at 4°C.

Publication protocol

For Western blot analysis, nuclear and cytoplasmic fractions were generated using the CelLytic NuCLEAR Extraction kit (Cat#NXTRACT Sigma-Aldrich,). 48 hours after transfection, cells were washed with PBS, collected, and resuspended in 0.5 ml of hypotonic lysis buffer and incubated on ice for 15 minutes, 30 μl of 10% IGEPAL was added to the cell suspension. Cells were then vortexed and spun at 10,000 xg for 30 seconds at 4°C. The supernatant (cytoplasmic fraction) was decanted and saved. The nuclear fraction was generated by adding 70 μl of nuclear extraction buffer to the pellet, incubating on ice for 25 minutes, spun at 20,000 xg for 5 minutes at 4°C and the supernatant was saved (nuclear fraction). Molecular weights for wild-type and mutant proteins were estimated using Protein Molecular Weight calculator (http://bioinformatics.org/sms/prot_mw.html). The BenchMark Pre-stained Protein Ladder (Cat. #10748-010) (Life Technologies) was utilized as the protein molecular weight standard.

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