Fluorography of Gels Using PPO
Step Description SDS-PAGE Gel Agarose Gel
Agitation Use rotating platform. Use rotating platform.
Use glass dish with glass top. Use glass dish with glass top.
1 Fixation Fix gel for 30 minutes. No need to fix gel.
2 Dehydration 1st DMSO for 30 min.* Glacial AcCOOH for 15 min.#
2nd DMSO for 30 min.* Fresh Glacial AcCOOH for 15 min.#
Fresh Glacial AcCOOH for 15 min.#
3 Impregnation 20% PPO in DMSO for 1 hour. 15% PPO in AcCOOH for 45 min.
4 Precipitation dH2O for 30 minutes. dH2O for 30 minutes.
Use running dH20 or several changes of dH20 to remove DMSO or AcCOOH.
5 Drying Dry for 1 hour at 60°C. Dry for 30 minutes without heat.
Then dry for 1.5 hours at 50°C.
6 Exposure Wrap with plastic film, and place against pre-flashed film at -70°C.
*DMSO may be re-used 10 times. Make hash-mark on tape on bottle. After 10 uses, use fresh DMSO
for 2nd solution, and use 2nd solution as new 1st solution.
#AcCOOH is not re-used. Use a minimum volume (enough to cover gel). Discard after use.
PPO stocks can be used for about 20 uses, or until contaminated by water (PPO precipitates) or until
PPO is exhausted (poor results on film), whichever comes sooner.