Ron Lab


 

Preparing Nuclear and Cytoplasmic Extracts


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 β-glycerophosphate
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 EDTA Method


  • 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 5 min.
  • 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 eIF2α western.
  • Wash/resuspend pellet in 500μl buffer A.
  • Pellet at 1000rpm in swinging bucket rotor 5min.
  • Remove and discard sup.
  • Add 4 vol buffer C. (for a more concentrated extract use 2 vol 2X buffer C)
  • Vortex 15 min 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.)