Fractionated Interphase Egg Extract

2.4.1. Preparation of Cytosolic and Light Membrane Fractions

1. Crude interphase extract.

2. 2.5-mL Ultra-Clear centrifuge tubes.

3. TLS centrifuge and TLS-55 rotor (Beckman) or equivalent.

2.4.2. Preparation of Mitochondria-Rich Heavy Membrane Fraction

1. Mitochondrial buffer (MIB) concentrate: prepare fresh; the following makes approx 14.5 mL of 4X MIB:

b. 600 |L Adenosine 5'-diphosphate (from 100 mM stock).

g. 600 |L Ethylene glycol Ws(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA; from 0.5 M stock).

3. Beckman TL-100 tabletop ultracentrifuge.

4. TLS-55 swinging bucket rotor.

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