Semi-purification of COX-2
Buffer
A 500 ml
200 mM Tris, pH 8.0 10
ml 1M Tris pH 8.0
0.4% CHAPS 20
ml 10% CHAPS
0.1 mM EDTA 100
ml 0.5 M EDTA
H2O
ad 500 ml
Buffer B 500
ml
80 mM Tris, pH 7.2 40
ml 1M Tris
2 mM EDTA 2
ml 0.5 M EDTA
H2O
ad 500 ml
1.
Chill
ultracentrifuge tubes & rotor (Beckman, 60 TI)
2.
Spin
infected Sf-9 cells (50-70% infected – Trypan blue) down in Sorvall – 10 min
2000 rpm
3.
Wash
cell pellet once with cold PBS & spin again with same conditions
4.
Freeze
pellet at -80øC
5.
Add
antioxidant Diethyldithiocarbamate (DETC, f.c. 0.1 mM) and 100x protease
inhibitor (Sigma) to Buffer B
6.
Thaw
pellet on ice and take up in 30 ml of Buffer B
7.
Put
in ultracentrifuge tubes, weigh and balance within 0.05g
8.
Spin
at 4øC for 1h at 40000 rpm [
2; r = radius of rotor]
9.
Pour
off supernatant and discard
10.
Resuspend
pellet in 3 ml of Buffer A and homogenize with a glass pastel and mortel
11.
Place
homogenate in glass vial with a stir bar and take to cold room
12.
Stir
while adding 180 ml of CHAPS 10% to a f.c. of 1%
13.
Stir
for 60 min, then place in ultracentrifuge tube again and spin at 4øC for 45 min
at 40000 rpm
14.
The
supernatant should contain the solubelized COX-2 enzyme