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) or primuline (lipids can be visualized with UV light). Scrape lipid bands from TLC directly into glass tubes.

Bacteria/Yeast

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 a fresh leaf or isolated lipid into a glass tube (of 5-8 ml volume) with screw cap (e.g. Schott, # 233 175 11 59). Use a 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, first put the a 1 N methanolic HLC and internal standard (15:0) into glass tube and put your sample directly into the liquid.

2. Immediately add 1 ml of 1 N HCl (3 N methanolic HCl from Supelco, order # 3-3050; dilute 1 to 3 with methanol - store at 4 °C) in methanol with a glass pipette.

   You can stop here: Close the vial with a screw cap and store it at 4 °C for a few days up to some weeks. Transport at room temperature or send by surface mail.

   Add internal standard: 100 ml of working stock (5 µg), using a yellow tip (Pentadecanoic acid, 15:0, working stock: 50 mg/ml in methanol, 1:200 of super stock (Superstock: 10 mg/ml in methanol)). (Pentadecanoic acid from Sigma, order # P 6125, 1 g 9.20 Euro)

3. Incubate at 80°C for 20 min (lipids isolated from TLC plates, bacteria pellets, leaves, roots) or 90 min (seeds)

4. Wait until cold.
   Add 1 ml 0.9 % NaCl

   Add 1 ml hexane (FAMEs go into the hexane/upper phase), shake vigorously and centrifuge for 3 min @ 1000 rpm.

5. Take off the hexane phase with Pasteur pipette and put it into a fresh glass tube without screw cap. Concentrate the FAMEs in air stream to approx. 250 µl. Do not dry completely, because this will blow out short and medium chain FAME's (10:0, 12:0, 14:0). For FAMEs from big leaves or lipid rich samples, you can omit this concentration step.

6. Add hexane to a volume of ca. 250 µl hexane (yellow tip). Fill into GC tubes. The sample can be stored @ -20°C.
    

Reference:
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