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. 2008 Sep;148(1):304–315. doi: 10.1104/pp.108.123331

Figure 7.

Figure 7.

Purification of (His)6-ACBP6 recombinant protein and its interaction with PC. A, Purification of (His)6-ACBP6 recombinant protein. An SDS-PAGE gel shows the 18.9-kD (His)6-ACBP6 protein purified from E. coli at 3 h after isopropylthio-β-galactoside induction. M, Marker. Lane 1, Flow-through fraction; lane 2, washing fraction at pH 6.3; lanes 3 to 8, eluted fractions at pH 5.9; lanes 9 to 12, eluted fractions at pH 4.5. B, (His)6-ACBP6/lipid binding on filters. Various concentrations (0, 12.5, 25.0, and 32.5 μm) of lipids (PA, PC, and lysoPC) were spotted onto nitrocellulose and incubated with 1 μg mL−1 purified (His)6-ACBP6 protein. The (His)6-ACBP6/lipid binding was detected by immunoblotting with HRP-conjugated anti-(His)6 antibodies. C, Effect of PC acyl species on (His)6-ACBP6/lipid binding. Fifty micromolar lipid (PC, 16:0-PC, 18:0-PC, 18:1-PC, 18:2-PC, or DMPC) spotted onto nitrocellulose was incubated with 1 μg mL−1 purified (His)6-ACBP6 protein. The (His)6-ACBP6/lipid binding was detected by immunoblotting with HRP-conjugated anti-(His)6 antibodies.