Extraction and TLC Analysis of Mitochondrial Phospholipids

Lipid extraction of isolated mitochondria was performed according to published method (Bligh and Dyer, 1959). Briefly, a mixture of chloroform:methanol 1:2 (v/v) was added to 600 µg of mitochondria, well-vortexed and incubated on ice for 30 min. Then, 1.25 times (v/v) of each chloroform and water were added and after vigorous vortex, samples were centrifuged (1000 rpm, 5 min, room temperature). The organic phase was collected and solvents were evaporated by speed vac. Then, lipids were re-suspended in 40 µL of chloroform:methanol 1:1 (v/v) mixture and either stored at -20°C for subsequent analysis or immediately used.

Phospholipid classes from the total lipid extract were separated by thin-layer chromatography (TLC) using glass plates of pre-coated 0.25 mm silica gel with fluorescent indicator UV254. Prior to separation, the silica plates were washed with a mixture of chloroform:methanol 1:1 (v/v) and dried for 15 min. Then, plates were sprayed with a solution of boric acid in ethanol (2.3% w/v) and dried in an oven at 100°C for 15 min. Next, 40 µL of sample of total lipid extract or 3-5 µL of PL standards were applied to the plate. The spots were air dried and developed in a solvent mixture of chloroform/ethanol/water/trimethylamine (30:35:7:35, v/v/v/v). After complete migration and eluent evaporation, the TLC plates were sprinkled with molybdenum blue solution. Identification of the different classes of PLs was carried out by comparison with the migration behavior of phospholipid standards.

Bligh, E.G., and W.J. Dyer. 1959. A RAPID METHOD OF TOTAL LIPID EXTRACTION AND PURIFICATION. Canadian Journal of Biochemistry and Physiology. 37:911-917.

Mitochondria Phospholipids Extraction

(Bligh and Dyer Method;1959)

1) For each sample take 600µg mitos (determined by protein measurement) or 500µg mitos (determined by phospholipids) and resuspend in a total volume of 600µl SEM buffer

2) For 600 µl sample add 2250 µl Chloroform:Methanol (1:2) (ratio volume is 1 sample 3.75 ChCl3:MeOH) and vortex 3min

3) Keep in 30min on ice

4) Add 1.25x CHCl3 (750ul CHCl3 for 600 µl sample) and vortex well

5) Add 1.25x ddH2O (750ul H2O for 600 µl sample) and vortex well

6) Centrifuge 5min, 1000rpm, RT to give a two-phase system (aqueous top, organic bottom)

7) Recover the bottom phase with Pasteur pipette through the upper phase with gentle positive-pressure (i.e., gentle bubbling) so that the upper phase does not get into the pipette tip.

8) Centrifuge again the organic recovered phase as in 6) and keep organic phase

9) Evaporate liquid (speedvac)

10)Resuspend pellet in 42µl Chloroform:methanol (1:1)

Separation of Mitochondria Phospholipids Classes by TLC

1) Silica Gel TLC-plates are washed prior to separation with a solution of CHCl3:MeOH (1:1 v/v) (~50mL total) until 1cm from top and dried in the hotte for 15min.

2) Plates are sprayed with a solution of boric acid in ethanol (2.3% m/v) and dried in an oven at 100°C for 15 min. (A cyclic boric acid derivative fixes the hydroxyl groups, and the relative stability of the complexes determines the separation possible. Di- and monoacylglycerols isomerise easily in acidic solution (and on silica gel), even in the cold, thus, they should be used as soon as possible for derivatization or further chromatography.)

3) Apply the samples in the TLC a. Standards 10µl b. Samples according to protein measurement: 1/3 and 2/3 (=200µg and 400µg) c. Samples according to phospholipid measurement: 1/3 and 2/3 (=160µg and 260µg)

4) Develop TLC in a glass chamber filled up with solvent mixture chloroform/ethanol/water/trimethylamine (30:35:7:35, v/v/v/v) (app. 2h)

5) Dry the plate on the hot plate for 5 min. (Please note: 5 min may be too long if the temperature is over 100C)

Spray molybdenum blue solution onto the plate until you see blue lipid bands

Protocol Courtesy of Professor Doron Rapaport, University of Tuebingen