220.127.116.11. Solution of radioactive ecdysone (RIA tracer solution)
The stability of
ecdysone in aqueous solution is limited. As this solution should be used within
4-6 weeks, it is necessary to estimate the volume needed for each assay series.
100 µl of this solution is required per sample, and the radioactivity should be
5,000-6,000 cpm/100 µl tritiated ecdysone ([23,24-3H(N)]ecdysone, Perkin Elmer NEN; NET621050UC; 1.85 MBq; 1.85-4.07 TBq/mmol). The
solution should be stored at 4oC.
1. As tritiated ecdysone comes as 50 µCi in 0.5 ml ethanol, calculate the volume to be used in an assay series.
2. Transfer to 5 ml disposable glass test tube and evaporated to dryness by vacuum centrifugation or under a N2-stream.
3. Dissolve the radioactive ecdysone in the appropriate volume of 0.1 M phosphate buffer.
4. Transfer to a screw cap glass vial.
5. Keep this vial on an orbital shaker for 24 h at 4oC.
6. Test radioactivity level by liquid scintillation counting of 100 µl aliquots.
This should give 5,000-6,000 cpm for 100 µl.
7. If necessary, adjust radioactivity level by adding phosphate buffer or tracer.