Sport and Exercise Sciences Research Institute
Standard Operating Procedure
EPR Protocol for Ascorbyl Radical using DMSO ( considered Hazardous)
(SS/SOP0016)
1. Please see relevant COSHH forms and sign that you have read these before starting
the assay
2. Switch analyser and water pump on 24 hours prior to use.
3. Thaw sample.
4. Open Bruker WinEPR acquisition software.
5. Open previous spectra for the particular radical under investigation (i.e. if looking at
ascorbyl open a previous ascorbyl trace).
6. Enter microwave bridge control (MW).
7. Select calibrated in interactive spectrometer control (I).
8. Select tune (operating mode) and click upwards arrow for auto tune.
9. Stand by, unlevelled and uncalibrated should turn green; the AFC and diode should be
green; and the attenuation should be 10 dB (manually adjust if necessary) and the
power 20 nW.
10. Flush cavity with saline then place fresh saline into the cavity and click run.
11. Once complete clean cavity with saline (ensure all is removed by flushing air
through).
12. Mix 1 ml of serum (refer to Risk assessment SS0005) with 1 ml of DMSO thoroughly
and take the resultant liquid into a syringe and flush through the EPR cavity.
13. Ensure all is still green and power etc is correct (if not select fine tune).
14. Click run.
15. Once complete save spectra by clicking file>save as.
16. Clean cavity once complete and continue with next samples.
17. To analyse click WinEPR icon on toolbar of the file.
18. Select WinEPR system then 1D-processing.
19. Select 1D-processing and then filter.
20. Select Conv. Point of 7 (ensure this is consistent for all images) and click ok.
21. Select display and then distance.
22. Move cursor to the tip of the peak near 3495 G (approximately) and click to fix the
line.
23. Then move to the trough of the peak and record the +/- val.
24. This is an arbitrary unit of measurement not a concentration.
25. Repeat for the next peak and average the two.