9. 40 ml Lysis buffer: 50 mM Na 2 HPO 4 , pH 7.6, 1 mM MgCl 2 ,
5 mM 2-mercapthoethanol.
10. 5 μl benzonase nuclease (10 U μl
À1 ), 5 μl lysozyme
(10 mg ml
À1 ).
11. 2 M urea, 1% (v/v) Triton X-100 (10 ml each).
12. 10 ml IB-lysis buffer: 6 M guanidinium–HCl, 0.035% (v/v)
2-mercaptoethanol.
13. Stirrer, 500 ml beaker.
14. 500 ml refolding buffer (RF): 50 mM HEPES, pH 7.6, 1 mM
tris(2-carboxyethyl)phosphine (TCEP), 0.2% (w/v) lauryldimethylamine N-oxide (LDAO), 1 mM MgCl 2 .
15. Fractogel EMD chelate (Merck), coupled with CoCl 2 (see
Note 1).
16. 15 ml and 50 ml plastic tubes.
17. 150 ml PSY washing buffer: 50 mM Tris, pH 7.6, 300 mM
NaCl, 1 mM TCEP, 1 mM MgCl 2 , 0.035 (w/v) % LDAO.
18. 50 ml PSY elution buffer: 50 mM Tris, pH 7.6, 150 mM NaCl,
1 mM TCEP, 0.035 (w/v) % LDAO, 100 mM EDTA.
19. 1 L PSY dialysis buffer: 50 mM Tris, pH 7.6, 600 mM NaCl,
1 mM TCEP, 0.035% (w/v) LDAO.
20. Standard regenerated cellulose dialysis membrane with 15 kD
cutoff (see Note 2).
21. Liquid nitrogen.
2.3 PSY Activity
Assay
1. 20 mg phosphatidylcholine (e.g., from soybean, Type II-S).
2. 50 ml liposomes incubation buffer: 1 mM DTE, 10 mM
MgCl 2 , 100 mM Tris–HCl, pH 7.2.
3. 2 ml methanol–chloroform (1:2, v/v).
4. 15 ml reaction tubes.
5. French press, sonicator, ice.
6. 10 ml PSY reaction buffer: 100 mM Tris–HCl, pH 7.6, 0.08%
(v/v) Tween 80, 20% (v/v) glycerol, 2 mM MnCl 2, 10 mM
MgCl 2, 1 mM TCEP, 600 mM NaCl (see Note 3).
7. [114
C]-IPP, triammonium salt in ethanol: 0.15 N NH 4 OH
(1:1) with 50–60 mCi mmol
À1
.
8. 1 mg ml
À1 (3.365 mM) DMAPP and IPP (triammonium salt)
in ethanol: 0.15 N NH 4 OH (1:1).
9. 50 μl 160 mM EDTA.
10. Butanol, 1 M MgCl 2 in methanol, heptane.
11. Incubator for reaction tubes at 20
C, table centrifuge.
12. Scintillation counter, scintillation cocktail, and scintillation
vials.
44
Maurizio Camagna and Ralf Welsch
5 mM 2-mercapthoethanol.
10. 5 μl benzonase nuclease (10 U μl
À1 ), 5 μl lysozyme
(10 mg ml
À1 ).
11. 2 M urea, 1% (v/v) Triton X-100 (10 ml each).
12. 10 ml IB-lysis buffer: 6 M guanidinium–HCl, 0.035% (v/v)
2-mercaptoethanol.
13. Stirrer, 500 ml beaker.
14. 500 ml refolding buffer (RF): 50 mM HEPES, pH 7.6, 1 mM
tris(2-carboxyethyl)phosphine (TCEP), 0.2% (w/v) lauryldimethylamine N-oxide (LDAO), 1 mM MgCl 2 .
15. Fractogel EMD chelate (Merck), coupled with CoCl 2 (see
Note 1).
16. 15 ml and 50 ml plastic tubes.
17. 150 ml PSY washing buffer: 50 mM Tris, pH 7.6, 300 mM
NaCl, 1 mM TCEP, 1 mM MgCl 2 , 0.035 (w/v) % LDAO.
18. 50 ml PSY elution buffer: 50 mM Tris, pH 7.6, 150 mM NaCl,
1 mM TCEP, 0.035 (w/v) % LDAO, 100 mM EDTA.
19. 1 L PSY dialysis buffer: 50 mM Tris, pH 7.6, 600 mM NaCl,
1 mM TCEP, 0.035% (w/v) LDAO.
20. Standard regenerated cellulose dialysis membrane with 15 kD
cutoff (see Note 2).
21. Liquid nitrogen.
2.3 PSY Activity
Assay
1. 20 mg phosphatidylcholine (e.g., from soybean, Type II-S).
2. 50 ml liposomes incubation buffer: 1 mM DTE, 10 mM
MgCl 2 , 100 mM Tris–HCl, pH 7.2.
3. 2 ml methanol–chloroform (1:2, v/v).
4. 15 ml reaction tubes.
5. French press, sonicator, ice.
6. 10 ml PSY reaction buffer: 100 mM Tris–HCl, pH 7.6, 0.08%
(v/v) Tween 80, 20% (v/v) glycerol, 2 mM MnCl 2, 10 mM
MgCl 2, 1 mM TCEP, 600 mM NaCl (see Note 3).
7. [114
C]-IPP, triammonium salt in ethanol: 0.15 N NH 4 OH
(1:1) with 50–60 mCi mmol
À1
.
8. 1 mg ml
À1 (3.365 mM) DMAPP and IPP (triammonium salt)
in ethanol: 0.15 N NH 4 OH (1:1).
9. 50 μl 160 mM EDTA.
10. Butanol, 1 M MgCl 2 in methanol, heptane.
11. Incubator for reaction tubes at 20
C, table centrifuge.
12. Scintillation counter, scintillation cocktail, and scintillation
vials.
44
Maurizio Camagna and Ralf Welsch