Polar lipids are generally extracted from dry cell material using chloroform:methanol:0.3% NaCl (1:2:0.8 v/v/v). This may be carried out by adding 9.5 ml of this mixture to 100 mg of freeze dried cells, or by adding a suitable amount of chloroform, methanol and 0.3% NaCl to the cell material, or to the aqueous methanolic phase remaining from the lipoquinone extraction.
- The aqueous methanolic phase (4 ml total volume), together with the cell material from the lipoquinone analysis, is diluted with 5.5 ml of Chloroform:Methanol (2.5:3.0 v/v) to give a chloroform, methanol, 0.3% NaCl (1:2:0.8 v/v/v) mixture.
- The mixture is placed in a 15 ml bottle with a teflon lined screw cap (check that the magnetic stirrer is in the bottle), gassed briefly with nitrogen, sealed and heated for 15 min at 80 C (with occasional shaking).
- Allow the mixture to cool to room temperature on a magnetic stirrer. Check that the mixture is homogeneous, the presence of excess hexane will cause phase separation and may be overcome by adding a small amount of methanol until a homogeneous mixture is obtained.
- The cell debris is removed by centrifugation in glass centrifuge tubes, at 3000 rpm for 5-10 min, and the supernatant decanted into 5 ml of chloroform:0.3% NaCl (1:1 v/v). The latter mixture (2.5 ml chloroform and 2.5 ml of 0.3% NaCl) may be placed in the extraction buffer bottles after they have been washed with distilled water.
- After brief mixing, the biphasic mixture is centrifuged in glass centrifuge tubes (3000 rpm for 5 min) and the lower, chloroform phase collected with a pasteur pipet. Take care not to remove the flocculent protein layer or the aqueous phase.
- The chloroform phase, containing the polar lipids is taken to dryness under a stream of nitrogen (when using a heating block or a water bath to accelerate drying, do not exceed 40 C).
- The dried lipid material is then re-dissolved in 250 ul of chloroform:methanol (2:1 v/v) and transferred toa small glass bottle or ampule and may be stored for periods of several months at temperatures of -20 C or lower.
- The lipid solution is used to spot thin layer plates; the lipids being placed in the bottom left-hand corner of each plate (Fig 4-A). Glass or aluminum plates are used because many detection reactions require the plates to be heated.
- Run the first dimension with the spotted lipid on the lower right hand corner. After drying, run the plates in the second dimension with the lipid in the lower left hand corner.
- Develop the plates in two dimensions using, in the first dimension chloroform:methanol:water (65:25:4 v/v/v), and in the second dimension, chloroform:methanol:acetic acid:water (80:12:15:4 v/v/v/v). Between the two dimensions, the plates should be dried at room temperature for about 20-30 min.
- Lipid functional groups are identified using spray reagents specific for phosphate (Zinzadze), alpha-glycols (periodate-Schiff), and sugars (alpha-naphthol/H2SO4, anisaldehyde/H2SO4), free amino groups (ninhydrin), quaternary nitrogen (Dragendorff), and primary and secondary amines (chlorine and starch/iodide). Identification of the various lipids is carried out on the basis of staining reaction and Rf values. All spraying must be carried out in a fume hood using sufficient ventilation, since all lipid spray reagents are toxic.