Planar fluid lipid bilayers were prepared as previously described (Brian and McConnell, 1984 (link); Grakoui et al., 1999 (link); Carrasco et al., 2004 (link)). In brief, Ni-NTA–containing lipid bilayers were prepared using 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-[N(5-amino-1-carboxypentyl) iminodiacetic acid]-succinyl (nickel salt; DOGS–Ni-NTA; Avanti Polar Lipids, Inc.) in a 9:1 DOPC/DOGS–Ni-NTA ratio. Unilamellar vesicles were formed by sonication of the mixed lipids and clarified by ultracentrifugation and filtering. Glass coverslips were cleaned by Nanostrip (Cyantek), washed, and dried. Lipid bilayers were prepared from 0.1-mM lipid unilamellar vesicle solutions on the coverslip attached to the bottom of Labtek chambers (Thermo Fisher Scientific).
NIP or pNP conjugated to the peptide ASTGKTASACTSGASSTGSHis12 (NIP1-His12 or pNP1-His12) and NIP1-His12 and pNP1-His12 coupled to Hylight647 through the cysteine residue were purchased from Anaspec. The hapten–peptide conjugates were HPLC purified and verified by mass spectroscopy with >95% purity. The hapten-coupled peptides were attached to the Ni-NTA–containing lipid bilayer by incubating haptenated peptides (10 or 50 nM) with the bilayer for 20 min at room temperature (RT). Where indicated in the figures, mouse ICAM-1/huFc chimera protein with a 10-nM His12 tag (R&D Systems) was also attached to the lipid bilayers. After washing, the antigen-containing lipid bilayers were used in TIRF imaging. The amount of antigen bound to the bilayer was quantified by titration of the Hylight647-conjugated peptides to resolve single molecules. The concentration of the peptide attached to the bilayer was calculated by a function of where C is the concentration (number of molecules per square micrometer), N is the number of Hylight647-conjugated peptide molecules counted at a single-molecule resolution per square micrometer, and D is the titration rate. In our experimental system, incubating bilayers with a 10-nM haptenated peptide solution resulted in bilayers containing ∼25 molecules/µm2, and incubating with a 50-nM solution resulted in a bilayer containing 100 molecules/µm2 in the planar lipid bilayer.
NIP or pNP conjugated to the peptide ASTGKTASACTSGASSTGSHis12 (NIP1-His12 or pNP1-His12) and NIP1-His12 and pNP1-His12 coupled to Hylight647 through the cysteine residue were purchased from Anaspec. The hapten–peptide conjugates were HPLC purified and verified by mass spectroscopy with >95% purity. The hapten-coupled peptides were attached to the Ni-NTA–containing lipid bilayer by incubating haptenated peptides (10 or 50 nM) with the bilayer for 20 min at room temperature (RT). Where indicated in the figures, mouse ICAM-1/huFc chimera protein with a 10-nM His12 tag (R&D Systems) was also attached to the lipid bilayers. After washing, the antigen-containing lipid bilayers were used in TIRF imaging. The amount of antigen bound to the bilayer was quantified by titration of the Hylight647-conjugated peptides to resolve single molecules. The concentration of the peptide attached to the bilayer was calculated by a function of where C is the concentration (number of molecules per square micrometer), N is the number of Hylight647-conjugated peptide molecules counted at a single-molecule resolution per square micrometer, and D is the titration rate. In our experimental system, incubating bilayers with a 10-nM haptenated peptide solution resulted in bilayers containing ∼25 molecules/µm2, and incubating with a 50-nM solution resulted in a bilayer containing 100 molecules/µm2 in the planar lipid bilayer.
