3.5.1. Purifying and preparing radioactive methionine for use in assay
Just prior to use, the labelled methionine will have to be washed to remove any unbound tritium, a source of contaminating “noise”.
1. Dilute 100 μCi of L-[methyl-3H]-methionine (>97 %, 1 mCi/ml, 70-85 Ci/mmol; PerkinElmer NET061X250UC) in insect saline to a final volume of 100 μl in a 1.5-ml microcentrifuge tube.
2. Add 500 μl of isooctane (2,2,4-trimethylpentane, anhydrous, 99.8 %; Sigma-Aldrich 360066) to the methionine.
3. Mix well by vortexing for 1 min.
4. Centrifuge at 10,000 x g for 10 min.
5. Using a pipettor, remove the isooctane (i.e. upper) layer, avoiding to touch the lower methionine/saline layer.
6. Repeat the wash steps 2-5 two more times.
7. After the third wash, transfer 100 μl of isooctane to a scintillation vial to determine how much contaminate remains.
7.1. Evaporate the isooctane in the
vial to near-dryness under nitrogen.
7.2. Add 3 ml of scintillation fluid with a high efficiency for tritium (e.g. ScintiSafe Econo F, Fisher Scientific) to the vial.
7.3. Vortex for 1 min.
7.4. Determine the radioactivity of the isooctane in a scintillation counter.
7.5. If the background count is sufficiently low (i.e., <1000 dpm), the methionine is ready for use.
7.6. If the background count is >1000 dpm, repeat the three wash cycle (steps 2-5).
7.7. If the cleaning process does not reduce the background dpm under 2,000 within two wash cycles, incubate the methionine in 500 μl of isooctane overnight (or longer) to remove more of the degraded isotope.
8. Evaporate any of the isooctane remaining in the vial with nitrogen gas.
9. For ten samples and a negative control, dilute 65 μl of the methionine solution in 1235 μl of Grace’s insect medium (from commercial supplier, e.g. Sigma-Aldrich) that is free of L-methionine.
This should provide a final (recommended) concentration of 100 μM methionine per incubation, with a specific activity of 5 μCi/100 μl. Higher concentrations will ensure that radioactive methionine is incorporated into any JH synthesized, but will also increase the background counts.