Cytoplasm and nuclear protein extraction: Difference between revisions
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Resuspend pellet in 3-5mL of Cytoplasm extraction Buffer. Put into 15mL glass pestle and crush 25-30 times on ice. Spin down at 2000rpm for 5min at 4∞C. Remove supernatant and transfer to microcentrifuge tube. Centrifuge at 14,000rpm for 15minute at 4∞C. Remove supernatant. This is the cytoplasmic protein solution. | Resuspend pellet in 3-5mL of Cytoplasm extraction Buffer. Put into 15mL glass pestle and crush 25-30 times on ice. Spin down at 2000rpm for 5min at 4∞C. Remove supernatant and transfer to microcentrifuge tube. Centrifuge at 14,000rpm for 15minute at 4∞C. Remove supernatant. This is the cytoplasmic protein solution. | ||
=='''Cleaning the pellet'''== | |||
#'''.25M sucrose buffer (10mL)''' | |||
#*.855g sucrose | |||
#*100uL MgCl2 (1M) | |||
#*100uL Hepes (1M pH7.5) | |||
#*water to 10mL | |||
#'''.35M sucrose buffer (10mL) | |||
#*1.2g sucrose | |||
#*5uL MgCl2 (1M) | |||
#*100uL Hepes (1M pH7.5) | |||
#*water to 10mL | |||
Add 3mL of 0.35M sucrose buffer to 14mL round bottom centrifuge tube. Resuspend pellet in 3mL 0.25M sucrose buffer. Tilt centrifuge tube and carefully pipet resuspended pellet in a layer over the 0.35M sucrose buffer. Centrifuge at 4∞C for 5min at 1430Xg. Aspirate supernatant. | |||
=='''Extracting nuclear proteins'''== | |||
#'''Nuclear lysis buffer''' | |||
#*10mM Hepes or Tris (pH 7.5) | |||
#*500mM NaCl | |||
#*1% Triton-X100 | |||
#*10% glycerol | |||
#*1mM NaPPi | |||
#*1ug/mL pepstatin | |||
#*1ug/mL aprotinin | |||
#*1ug/mL leupeptin | |||
#*1mM NaVO4 | |||
#*1mM NaF | |||
#*1mM PMSF | |||
#*water to 50 mL | |||
Resuspend the pellet in nuclear lysis buffer. Sonicate pellet for 30 seconds with a 1 minute rest 5-8 times at power setting 5. Centrifuge pellet at 14,000 rpm for 15 minutes at 4∞C. Remove and save supernatant. This is the nuclear protein solution. | |||
Revision as of 12:35, 21 August 2006
Harvest cells and wash with 1XPBS
Cytoplasm extraction
- Cytoplasm extraction buffer
- 10mM Hepes or Tris (pH7.5)
- 40mM KCL
- 2mMMgCl2
- 10% glycerol
- 1mM NaPPi
- 1ug/mL pepstatin
- 1ug/mL aprotinin
- 1ug/mL leupeptin
- 1mM NaVO4
- 1mM NaF
- 1mM PMSF
- water to 50 mL
Resuspend pellet in 3-5mL of Cytoplasm extraction Buffer. Put into 15mL glass pestle and crush 25-30 times on ice. Spin down at 2000rpm for 5min at 4∞C. Remove supernatant and transfer to microcentrifuge tube. Centrifuge at 14,000rpm for 15minute at 4∞C. Remove supernatant. This is the cytoplasmic protein solution.
Cleaning the pellet
- .25M sucrose buffer (10mL)
- .855g sucrose
- 100uL MgCl2 (1M)
- 100uL Hepes (1M pH7.5)
- water to 10mL
- .35M sucrose buffer (10mL)
- 1.2g sucrose
- 5uL MgCl2 (1M)
- 100uL Hepes (1M pH7.5)
- water to 10mL
Add 3mL of 0.35M sucrose buffer to 14mL round bottom centrifuge tube. Resuspend pellet in 3mL 0.25M sucrose buffer. Tilt centrifuge tube and carefully pipet resuspended pellet in a layer over the 0.35M sucrose buffer. Centrifuge at 4∞C for 5min at 1430Xg. Aspirate supernatant.
Extracting nuclear proteins
- Nuclear lysis buffer
- 10mM Hepes or Tris (pH 7.5)
- 500mM NaCl
- 1% Triton-X100
- 10% glycerol
- 1mM NaPPi
- 1ug/mL pepstatin
- 1ug/mL aprotinin
- 1ug/mL leupeptin
- 1mM NaVO4
- 1mM NaF
- 1mM PMSF
- water to 50 mL
Resuspend the pellet in nuclear lysis buffer. Sonicate pellet for 30 seconds with a 1 minute rest 5-8 times at power setting 5. Centrifuge pellet at 14,000 rpm for 15 minutes at 4∞C. Remove and save supernatant. This is the nuclear protein solution.
