Separation of Mitochondrial Membrane proteins from Soluble and Membrane-Bound proteins by Salt extraction and Carbonate wash

Liver mitochondria isolated by standard methods are ‘contaminated’ by a small number of lysosomes, peroxisomes and autophagic vacuoles, which in this protocol will rupture, leading to release of their content of proteolytic activities. Therefore you should bear this in mind performing the protocol without protease inhibitors!

1. Rat liver mitochondria are suspended in 150 mM KCl, 10 mM Hepes-K, pH 7.5 at a concentration of 1-5 mg protein/ml. Add desired protease inhibitors (e.g. protease inhibitor cocktail tablets (Sigma or Roche) plus EGTA and phenylmethylsulphonylfluoride). The suspension is sonicated at high intensity with a tip sonicator until the turbidity of the suspension no longer decreases indicating that al mitochondria are broken into submitochondrial particles. Usually it takes 5-10 min. Use constant cooling in ice/water while sonicating.

2. Spin the samples at 100 000 g for 60 min at +4 C. Collect the supernatant (S1) and the pellet.

3. Resuspend the pellet in 100 mM Na₂CO₃ at pH 11 (adjusted with NaOH) and keep on ice/water mixture. Use a glass potter for effective resuspension of the submitochondrial particles. Sonicate for 1 min at high intensity. Incubate for 30 min and sonicate again for 1 min.

4. Spin the samples at 100 000 g for 60 min at +4 C. Collect the supernatant (S2) and the pellet.

5. Resuspend the pellet at 1-5 mg protein/ml in 10 mM Hepes/Tris, pH 7.4.

The pellet is now ready for acetone precipitation to remove lipids, and the supernatants S1 and S2 for TCA precipitation to remove salt.

© 2003 Ove, Linn & Julius