Nuclear Extract Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Cells were lysed in a buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4,1 mM NaF, 2 mM imidazole, and a cocktail of protease inhibitors.	- Nuclear Extract kit was used accordingly to the manufacturer’s instructions.

Upstream tips
- Cells were lysed in a buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4,1 mM NaF, 2 mM imidazole, and a cocktail of protease inhibitors.
Protocol tips
- Nuclear Extract kit was used accordingly to the manufacturer’s instructions.

Publication protocol

Cells were lysed in a buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4,1 mM NaF, 2 mM imidazole, and a cocktail of protease inhibitors (Complete, Mini, EDTA-free, Roche, Basel, Switzerland). For the enrichment of nuclear proteins, Active Motif Nuclear Extract kit (Active Motif Europe SA, Brussels, Belgium) was used accordingly to the manufacturer’s instructions. Lysates were sonicated for 5 s, and centrifuged at 12,000× g for 10 min at 4 °C. After quantification with the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA), the equivalent of 30 μg of proteins, was mixed with Laemmli buffer 4× (250 mM Tris-HCl, pH 6.8, 8% SDS, 40% glycerol, 0.4 M DTT) and warmed at 95 °C for 5 min. Protein lysates were separated by 10–15% SDS-gel electrophoresis, and transferred onto Immobilon-P membranes (Merck Millipore, Darmstadt, Germany). Non-specific binding sites were blocked with an incubation of 1 h at RT in 5 % non-fat dried milk in TBST solution. The blots were then incubated at 4 °C overnight with the following antibodies diluted in a 5% BSA TBST solution. After washing, blots were exposed for 1 h at RT to HRP-conjugated secondary antibodies. Immunoreactivity was visualized with Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore).

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Manufacturer protocol

Download the product protocol from Active Motif for Nuclear Extract Kit below.

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