Reaction volume 100 ul
5 ul NADPH (100 mM)
x ul cell extract so you get 50 % conversion
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:
7,5 ml Aceton
add Methylenchloride to 100 ml
mix and fill in the chromatography chamber
start a prerun with whatmanpaper
Loading of silica plates
Mix samples and load 10 ul
Let dry and load the other 10 ul
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.