Cell nuclear protein preparation protocol for western blot
发布日期:2015-03-04阅读次数:4739
Isolation of nuclei from cells.
Reagents
Reagents
Buffer A
20 mM Tris pH 7.5–8.0
100 mM NaCl
300 mM sucrose
3 mM MgCl2
Buffer A contains sucrose and should be kept frozen at -20°C.
Buffer B
20 mM Tris pH 8.0
100 mM NaCl
2 mM EDTA pH 8.0
Keep at 4°C.
Buffer C
20 mM Tris pH 8.0
100 mM NaCl
2 mM EDTA pH 8.0
2% SDS
Keep at room temperature.
Method
- Prepare 1 ml of buffer A with added cocktail of usual protease inhibitors from frozen stock and store on ice.
- Add 500 µl buffer A per large petri dish on ice and scrape thoroughly. Leave on ice for 10 min.
- Centrifuge at 4°C at 3,000 rpm for 10 min.
- Remove supernatant and retain. This will contain everything except nuclei.
- On ice, resuspend the pellet in 374 µl buffer B and add 26 µl of 4.6 M NaCl to give 300 mM NaCl (high salt helps lyse membranes and forces DNA into solution).
- Homogenize with 20 full strokes in Dounce or glass homogenizer on ice.
- Leave on ice for 30 min.
- Centrifuge at 24,000 g for 20 min at 4°C.
- Aliquot supernatant, remove 10 µl for protein quantification (ab102536) and store at -70°C
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