Subcellular fractionation

Subcellular fractionation

Hypotonic lysis buffer:

250mM Sucrose,

20mM HEPES PH 7.4,

10mM KCl,

1.5mM MgCl₂,

1mM EDTA,

1mM EGTA,

1mM DTT,

protease/phosphatase inhibitors.

Procedure

All operations are at 4 ℃ or on ice except noted

1.    Decant cell culture medium, wash cells with room temperature PBS twice.

2.    Put cell culture plate on ice, add 1ml ice-cold hypotonic buffer, keep the cell culture plate on ice for 20 min.

3.    Scrape cell culture plates and collect the cell lysates.

4.    Then pass lysates through a 30-gauge needle 10 times by using a 1 ml syringe.

5.    Briefly centrifuge the sample by using a palm centrifuge, collect the supernatant and pass lysates through a 30 gauge needle 10 times.

6.    Centrifuge the sample at 14,000 g for 20 min. Collect the supernatant.

7.    Centrifuge the supernatant at 50,000 rpm (Beckman Coulter Type 70.i Fixed- Angle Rotor) for 2 hr. Collect the supernatant, which is the cytosolic fraction.

8.     Wash the pellet by adding 1ml hypotonic buffer to the pellet. Then re-centrifuge for 2 hr.

9.    The pellet is the membrane fraction, which contains plasma membrane, microsomes, and small vesicles.

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