Protocol: Fractionation of Membrane / Cytoplasmic and Nuclear Proteins

Protocol: Fractionation of Membrane / Cytoplasmic and Nuclear Proteins

Protocol 1 . Quick Fractionation

Materials

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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.

Your Expertise, Our Antibodies, Accelerated Discovery . USA Tel: 1.949.553.1900 Toll-free: 1.877.436.3839 International Tel: 886.3 .6208988

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Your Expertise, Our Antibodies, Accelerated Discovery. USA Tel: 1.949.553.1900 Toll-free: 1.877.436.3839 International Tel: 886.3 .6208988