Protocol for isolating both cytoplasmic and nuclear extracts: 10/29/02
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Solutions:
Harvest buffer:
10mM HEPES pH 7.9
50mM NaCl
0.5M Sucrose
0.1mM EDTA
0.5% Triton x 100
*freshly add to harvest buffer just before use:
1mM DTT
10mM tetrasodium pyrophosphate
100mM NaF
17.5 mM b-glycrophosphate
1mM PMSF
4 mg/ml Aprotinin
2 mg/ml Pepstatin A
Buffer A:
10mM HEPES pH 7.9
10mM KCL
0.1mM EDTA
0.1mM EGTA
*freshly add to buffer A just before use:
1mM DTT
1mM PMSF
4 mg/ml Aprotinin
2 mg/ml Pepstatin A
Buffer C:
10mM HEPES pH 7.9
500mM NaCl
0.1mM EDTA
0.1mM EGTA
0.1% IGEPAL(NP40)
*freshly add to buffer C just before use:
1mM DTT
1mM PMSF
4 mg/ml Aprotinin
2 mg/ml Pepstatin A
Ice cold PBS and PBS-1mM EDTAMethod:
• Place plates on ice.
• Wash 2X with cold PBS.
• Add 1ml PBS-EDTA and scrape cells. Transfer to 1.5ml tube.
• Pellet at 3000rpm 5 min.
• Resuspend in 250-500ml (at least 6 vol) Harvest buffer.
• Incubate on ice 5min.
• Pellet at 1000rpm in swinging bucket rotor for 10min to pellet nuclei.
• Transfer sup to new tube. For best results, clear at 14,000rpm for 15 min. &
transfer sup to new tube = Cytoplasmic/membrane extract.
Save for PERK or IRE IP or eIF2a western.
• Wash/resuspend pellet in 500ul buffer A.
• Pellet at 1000rpm in swinging bucket rotor 5min.
• Remove and discard sup.
• Add 4vol buffer C. (for a more concentrated extract use 2 vol 2X buffer C)
• Vortex 15min at 4C. (in cold room) Start on high speed vortex to loosen pellet,
then turn to medium.
• Pellet at 14,000rpm 10min 4C.
• Transfer sup to new tube. =Nuclear extract
If nuclear extract will be used for functional assays such as gel shift (EMSA),
then 10% glycerol should be added prior to freezing.
Measure protein concentrations of cytoplasmic and nuclear extracts.
Load ~40-50mg of nuclear extract for XBP , ATF4 and CHOP western (20mg is
enough for many cell types, but loading more is better.)
Load 25-40mg of cytoplasmic extract for P-eIF2alpha western.
Use 500-1500mg of cytoplasmic extract for PERK or IRE IP-western.
(The more the better.)