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Extraction of membrane proteins

Posted on Wednesday, June 07, 2006

Description
Membrane should be prepared.

Procedure
1. Add 1ml CHAPS extraction buffer containing protease inhibitors in membrane preparation (500 microgram).
2. Disperse the tissue by sonication (30sec bursts with 30 sec breaks).
3. Place in Sorvall at 4C, 12,000xg for 15 minutes.
4. Collect supernatent.
5. Add 1ml CHAPS extraction buffer again to the pellet. Mix well with syringe or pipette. Make sure to disperse the tissue.
6. Sorvall again at same conditions.
7. Pool supernatent with previous collection.
8. Repeat steps 5-7.
9. Dialyze the pooled supernatent to remove salts and detergents. Dailysis should be performed at 4C, changing cold distilled water with 6 changes every 2 hrs and one overnight change.
10. After dialysis, tranfer samples to fresh tube.

Recipes
1. CHAPS extraction buffer (0.5%) with protease inhibitors: Dissolve Benzamide (5mM), N-ethymalaimide (5mM), 6-aminohexanoic acid (0.1M) and CHAPS (0.5g) in 100ml sodium phosphate buffer.


Supplies
Dialysis tubing: cut-off siz m.w. 6,000 to 8,000

Tips


Submitted by: Asifa Ahmed

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