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Immobilized Phospholipid Chromatography 297
H 3 C CH 3
N
CH 3
H 3 C CH 3
N
CH 3
H 3 C CH 3 H 3 C CH 3 H 3 C
N N N
CH 3 CH 3 CH 3
H 3 C CH 3
N
CH 3
H 3 C CH 3
N
CH 3
O O
P
O
O
O O
P
O
O
O O
P
O
O
O O
P
O
O
O O
P
O
O
O O
P
O
O
O O
P
O
O
O
O
O
O
O
O
O
O
O
O O O
O O
O O O
O O
O
O
O
O
O
O
O
O
O
O
NH
O
NH
O
NH
O
NH
O
NH
O
NH
O
NH
CH 3
O
NH
O
NH
O
NH
O
NH
Si
O O
O
Si
O O
O
Si
O O
O
Si
O O
O
Si
O O
O
Si
O O
O
Si
O O
O
Si
O O
O
Si
O O
O
Si
O O
O
Si
O O
O
SiO 2
Fig. 1. General structure of phosphatidylcholine immobilized silica substrate. The
phosphatidylcholine is covalently bound to propylamine groups and the residual amines
are blocked with decanoic anhydride (Cl0 groups) followed by propionic anhydride
(C3 groups).
In this chapter, a protocol for the isolation of proteins with affinity to
membrane lipids is described using the immobilized phosphatidylcholine
column.
2. Materials
2.1. Chemicals and Reagents
1. Milli-Q water.
2. Tris base.
3. Sodium octanesulfonate.
4. Acetonitrile (CH 3 CN), HPLC grade.
5. Ethylene glycol.
6. 0.1 M phenylmethylsulfonylfluoride (PMSF) dissolved in isopropanol.
7. Trypsin solution: 10 μg/mL trypsin in sample buffer.
8. 0.1 M NaCl.
9. CaCl 2 .hexahydrate.
10. (NH4) 2 SO 4 .
11. Sample buffer: 25 mM CaCl 2 , 50 mM Tris–HCl, pH 7.6.
12. Tissue-homogenizing solution: 0.1 M NaCl in Milli-Q water.
13. 0.1 M solution of PMSF in isopropanol.