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TCA-ether extraction

This page is provided for the interested user. The method is a bit tricky to do, and uses a number of unpleasant chemicals; please make sure you comply with your local health and safety rules. If you are working in JIC, you will need to complete a COSHH assessment, and can get some help and guidance from us, or various groups in the department of Metabolic Biology who have used this method.

Method

  • Grind material (e.g. 500-600mg potato tuber) in liquid nitrogen in a pre-cooled mortar. When it's a fine powder, add 1500µl (= 2 lots of 750µl) 16% (w/v) TCA in ether, and mix it a bit with the pestle. If you've got the cooling right this will be sticky but not solid. Work in a fume-hood, and wear gloves.
  • Leave it 15 min on dry ice, covered with a paper towel to stop damp air/flies/dust from getting in.
  • Add 800µl aqueous TCA (16% w/v) containing 5mM EGTA, and for "problem" tissues 5mM EDTA, 20mM NaF (an acid solution containing NaF is effectively HF - be careful!). On addition this may spit - eye protection is essential. Pulp the frozen solution with the pestle - it should now go like mushy snow. Then let it thaw. When it has thawed, transfer it to a 2-mL microcentrifuge tube. The round-bottomed type with little clippy-bits on the lid are good for this volume. Leave it 2.5 to 3 hours on ice. Don't worry that the volumes add up to more than 2mL. Some of the ether will have evaporated, and you won't be able to remove all the liquid from the mortar.
  • Add a little water-saturated ether to replace any ether that has evaporated, and centrifuge 5min, 4°C, 10000g. Don't add too much; you may need to pipette out the lower of the two phases in the next step, and if there's no spare volume in the tube you won't be able to stick a pipette tip in without the top phase running down the side of the tube. Water-saturated ether is what you get if you add 50ml water to 500ml ether and shake (it's the top phase of the two that separate). Be careful when you pipette water-saturated ether not to pipette the water! If you suddenly find your aqueous phase has doubled in volume, that is probably what you have done.
  • Separate the phases and thow away the ether and the solid. How you do this is a matter of personal choice. Ether is supposed to be lighter than water, but at this stage there's so much TCA present, preferentially dissolving in the ether, that the ether might actually be heavier than the water. Although theoretically one ought to pipette ether with a glass pipette, Pasteur pipettes are not suitable for this step unless you have incredibly steady fingers; it's too difficult to avoid stirring up the sediment. Your final extract should not contain any of the solid. Amazingly some proteins come back to life after precipitation with TCA when they find themselves back in a neutral solution. They could disturb your measurements later.
  • Add more water-saturated ether, mix, and centrifuge. Throw away the ether.
  • Repeat step 6 until you've done it 4 times (3 may be enough). Neutralise the final aqueous phase with anything you like (traditional is 5M KOH, 1M Triethanolamine, of which you should not need more than 10µl). A good way to check the pH is to stir the solution a bit with a disposable plastic stirrer, and then use the stirrer to drip a tiny bit on narrow-range pH paper. Be very careful not to over-neutralise if you want to measure alkali-labile metabolites such as triose phosphates.