Phastsystem

Recombinant mouse CD1d was expressed and purified as previously
reported⁵³ . CD1d was
loaded with 3× molar excess of SLF (bovine brain, Avanti Polar Lipids;
dissolved at 5 mg ml⁻¹ in DMSO) in 100 mM Tris (pH 7.0) 100 mM
NaCl at 37 °C for 2 h. After lipid loading, excess lipid was removed by
ultrafiltration using Amicon filter cartridges (30-kDa molecular weight
cut-off). CD1d-SLF complexes were then incubated overnight at room temperature
with increasing concentration of either sphingomyelin (C24:1) (synthetic, Avanti
Polar Lipids) or α-GalCer. Lipids were dissolved in DMSO (5 mg
ml⁻¹) and incubated with CD1d in the presence of 0.01%
tyloxapol. As a control, CD1d-SLF complexes were incubated with only tyloxapol.
In a reversed experiment, CD1d was first loaded with 6× molar excess of
either sphingomyelin (10 mg ml⁻¹ in DMSO) or α-GalCer
(0.2 mg ml⁻¹ in vehicle) at room temperature overnight,
purified from excess lipid by ultrafiltration and incubated with 3× molar
excess of SLF for 1 h at 30 °C, which is the minimum dose and time
necessary for full loading of insect-cell-expressed mouse CD1d (data not shown).
Four microliters (2–4 μg) of the various CD1d-lipid-loading
products were analyzed using native IEF gel electrophoresis on pH 5–8
gels with a PhastSystem (GE Healthcare) and stained with Coomassie dye.

Aliquots of 10 μl purified mCD1d at a concentration of 20 μM were loaded overnight at room temperature in the presence of 6-times molar excess of each lipid. Sulfatide was dissolved in DMSO (5 mg/ml), and PA was dissolved in CHCL₃ (10 mg/ml). Loading was performed in the presence of 100 mM Tris-HCl (pH 7). After lipid loading, samples were centrifuged (14,000×g, 10 min), and 4 μl supernatant was used for isoelectric focusing (IEF) analysis. CD1d–lipid complexes were separated using precast gels (PhastGel 5–8 IEF) and the PhastSystem (GE Healthcare Biosciences, Piscataway, NJ). Staining of mCD1d was performed with Coomassie blue, and successful lipid loading was identified by a gel shift of the mCD1d band in relation to the control lanes.