fractionation of membrance cytoplasmic and nuclear proteins · c • confluent cell culture plates...
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Protocol: Fractionation of Membrane / Cytoplasmic and Nuclear Proteins
Protocol: Fractionation of Membrane / Cytoplasmic and Nuclear Proteins
Protocol 1 . Quick Fractionation
Materials
C • Confluent cell culture plates OJ
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Buffers • Triton X-100 lysis buffer: 50 mM Tris-HCI (pH 7.5), 0.5% Triton X-100, 137.5 mM NaCI, 10% Glycerol , 1 mM sodium vanadate, 50 mM sodium fluoride, 10
mM sodium pyrophosphate, 5 mM EOTA, and protease inhibitors (1mM PMSF, 10~g/ml aprotinin, 10~g/mlleupeptin).
• 2X SOS-PAGE sample buffer: 125mM Tris-HCI pH 6.8, 4% SOS, 20% glycerol , 10% beta-mercaptoethanol, 0.004% bromphenol blue.
Instruments
• Refrigerated microcentrifuge
Method
1. Lyse cells with 0.5% Triton X-100 lysis buffer. Incubate on ice for 15 min.
2. Separate insoluble nuclei by centrifugation at 3k rpm for 5 min at 4'C with a microcentrifuge Ooes this need a refrigerated microcentrifuge or standard microcentrifuge.
3. Transfer the supernatant (membrane/cytoplasmic fraction) into a new microcentrifuge tube.
4. Rinse the nuclear pellet with the lysis buffer once, then re-suspend in the lysis buffer. Pre-clear by centrifugation at 13,000 rpm for 15 min at 4 'c with a microcentrifuge. Transfer supernatant to a new tube.
5. Add an equal amount of 2X SOS-PAGE sample buffer to the tubes containing the nuclear and membrane/cytoplasmic fractions and boil both tubes for 10 min.
6. Successful fractionation can be verified by absence of ONA in the membrane/cytoplasmic-fraction by agarose gel electrophoresis. And likewise, the absence of beta-tubulin detected by Western blot indicates successful nuclear fractionation.
Protocol 2. Isolating both cytoplasmic/membrane and nuclear proteins
Materials
• Cell culture plates
• Cell scraper
Buffers
• Harvest Buffer: 10mM HEPES pH 7.9, 50mM NaCI, 0.5M sucrose, 0.1mM EOTA, 0.5% Triton X-100, and freshly added 1mM OTT, 10mM tetratsodium pyrophosphate, 10mM NaF, 17.5mM beta-glycrophosphate, 1 mM PMSF, 4~g/ml Aprotinin , 2~g/ml Pepstatin A.
• Buffer A: 10mM HEPES pH 7.9, 10mM KCI, 0.1mM EOTA, 0.1mM EGTA, and freshly added 1mM OTT, 1mM PMSF, 4~g/ml Aprotinin , 2~g/ml Pepstatin A.
• Buffer C: 10mM HEPES pH 7.9, 500mM NaCI, 0.1mM EOTA, 0.1mM EGTA, 0.1% NP-40, and freshly added 1mM OTT, 1mM PMSF, 4~g/ml Aprotinin, 2~ g/ml Pepstatin A.
• Ice-cold PBS and PBS-1mM EOTA.
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Protocol: Fractionation of Membrane / Cytoplasmic and Nuclear Proteins
Instruments
• Refrigerated microcentrifuge
• Microcentrifuge with swinging bucket rotor
• Vortex in cold room
Method
1. Place plates on ice.
2. Wash 2X with cold PBS.
3. Add 1.0 ml PBS-EDTA and scrape cells. Transfer to a microcentrifuge tube.
4. Pellet at 3k rpm for 5 min at 4'C.
5. Resuspend in cold harvest buffer (250-500~1).
6. Incubate on ice for 5 min.
7. Pellet at 1,000 rpm in swinging bucket rotor for 10 min to pellet nuclei.
8. Transfer the supernatant to a new tube and for best results, clear the supernatant at 14,000 rpm for 15 min and transfer to a new tube. This will contain the cytoplasmic and membrane proteins.
9. Wash and resuspend pellet from step 7 in 500~1 of Buffer A.
10. Pellet at 1000 rpm in a swinging bucket rotor. Discard supernatant.
11. Add 4 volumes of Buffer C (for a more concentrated extract, use 2 volumes of 2X Buffer C).
12. Vortex 15 min at 4°C, start on high speed vortex to loosen pellet, then turn to medium.
13. Pellet at 14,000 rpm for 10 min at 4°C.
14. Transfer the supernatant to new tube. This contains the nuclear extract.
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