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0.2 uM C14-Testosterone
add Tris-Citrate Buffer pH 6 to an endvolume of 100 ul
mix C14-Testosterone and Citrate Buffer and incubate it at 37 C
Incubate all together 10 min at 37 C
Stop reaction with 500 ul Methylenchloride
Vortex 15 sec
Centrifuge 2 min at 13000 rpm
Transfer the lower phase into a new tube
Speed vac 15 min (medium heat)
Elute in 20 ul Ethanol
Variations:
-Different volumes cell extract for finding out the 50% conversion
-Different concentration of testosterone: by using 0.2 uM C14 testosterone and cold testosterone. In that case fill in first the cold testosterone and speed vac 5 min to get rid of the ethanol.
-Different concentration of NADPH
-Different pH of Tris-Citrate Buffer
Preparation of the Chromatography step:
Put silica plate in the chamber and let it run for 45 min.
After running take it out of the chamber and let dry and lay a phosphorscreen on it.
Keep exposing for 2 days.