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Plant tissues:
For transmethylation you can use whole tissue (e.g. 1 small Arabidopsis leaf ca. 50 mg or roots 50 mg, leaf disk from tobacco, disc from potato tuber, 10 Arabidopsis seeds). For quantification you should determine fresh weight or size of leaf disc or count seeds. Put tissue directly into 1 M HCl in methanol (see below).

Lipids isolated by TLC:
Stain lipids on TLC plate with iodine vapor (use pasteur pipette filled with glass wool and iodine attached to a N2 stream to briefly stain the lipids. Don't saturate lipids with iodine, because polyunsaturated fatty acids may get covalently modified. Very important for plants, not so important for bacteria.). Scrape lipid bands from TLC directly into glass tubes.

Grow E. coli (or Rhodobacter, Synechococcus, ...) in 5-200 ml of  medium as usual. Harvest cells by centrifugating at ca. 5000 x g. (Optional: If desired, extract lipids secreted into the medium by extracting medium with chloroform/methanol (1:1)). Can freeze cell pellet in liquid N2 and store at -20 C or trans-methylate right away.

FAME Reaction:

  1. Put fresh leaf or isolated lipid into glass tube (of 5-8 ml volume) with screw cap (e.g. Schott, # 233 175 11 59). Use teflon septa in caps, not plastic or rubber (Schott, caps: # 29 240 08 06, teflon disc # 29 248 08 05). If you start with frozen plant material, put first methanolic HLC and internal standard (15:0) into glass tube and throw your sample directly into the liquid.
  2.  Immediately add 1 ml 1 N HCl in methanol (store at 4 C) with glass pipette.
    (3 N methanolic HCl from Supelco, order # 3-3050; dilute 1 to 3 with methanol)
    Can stop here. Close with screw cap and store at 4 °C for a few days up to some weeks. Can transport at room temperature or send by surface mail.
    Add internal standard: 100 ml of working stock (5 µg), use yellow tip.
    (Pentadecanoic acid from Sigma, order # P 6125, 1 g 9.20 Euro)
    pentadecanoic acid, 15:0, working stock: 50 mg/ml in methanol, 1:200 of super stock,
    Superstock: 10 mg/ml in methanol
  3.  Incubate at 80 C for     20 min (lipids isolated from TLC plates, bacteria pellets, leaves, roots)     90 min (seeds)
  4. wait until cold.
    + 1 ml 0.9 % NaCl
    + 1 ml hexane (FAME goes into hexane/upper phase)
    shake vigorously, centrifuge 3 min, 1000 rpm.
  5. Take off hexane phase with Pasteur pipette. Put into fresh glass tube without screw cap.
    Concentrate FAME in air stream down to ca. 250 µl. Do not dry completely, because this will blow out short and medium chain FAME's (10:0, 12:0, 14:0). For FAME from big leaves, can omit this concentration step.
  6.  Add hexane to a volume of ca. 250 ml hexane (yellow tip). Fill into GC tubes. Can store at - 20C.

Browse J, McCourt PJ, Somerville CR (1986) Fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue. Anal Biochem 152: 141-145

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