A BIACORE T200 instrument was used to determine interactions between purified MHC/peptide complexes and TCR molecules. TCR were immobilized on lipid bilayers coated on L1 chips by capture of their histidine tag. Lipid bilayers were produced by fusion of liposomes on the hydrophobic surface. Phosphatidylcholine (PC), cholesterol (C), dioleoyl Phosphatidylcholine (DOPC), L-α-phosphatidylethanolamine (PE), L-α-phosphatidylglycerol (PG), 1,2-dioleoyl-sn-glycero-3-[(n -(5-amino-1-carboxypentyl)imidodiacetic acid)succinyl](DOGS-NTA) were all from Avanti Polar Lipids*, and liposomes composition was 60:20:5:5:10 (PC:C:DOPC:PE:PG:DOGS-NTA) weight ratio. Liposomes were produced by extrusion at 1mg.ml−1 in PBS on 400nm polycarbonate disks(58 (link)). Streptag modified pMHC molecules were injected over the surface after stabilization of baseline in filtered and degassed PBS at a flow rate of 20-30 μl/min. To avoid regeneration of the surface between injections, kinetics studies were performed using the single cycle mode using an irrelevant TCR in the subtraction flow cell. Kd values, as well as on- and off-rates, were obtained by non-linear curve fitting of subtracted curves using the 1:1 Langmuir binding model using the BIAevaluation program (version 3.0.2).
Dogs nta
Manufactured by Avanti Polar Lipids
DOGS-NTA is a lipid compound used as a chelating lipid in liposome formulations. It is designed to incorporate nickel ions for the purification and immobilization of his-tagged proteins.
Automatically generated - may contain errors
Lab products found in correlation
Mini extruder, by Avanti Polar Lipids (1 mentions)
Polycarbonate filter, by Avanti Polar Lipids (1 mentions)
Polyvinyl alcohol (pva), by Thermo Fisher Scientific (1 mentions)
Phosphatidylcholine, by Avanti Polar Lipids (1 mentions)
Dioleoylphosphatidylcholine, by Avanti Polar Lipids (1 mentions)
L α phosphatidylethanolamine, by Avanti Polar Lipids (1 mentions)
L α phosphatidylglycerol, by Avanti Polar Lipids (1 mentions)
Anti his tag monoclonal antibody, by Merck Group (1 mentions)
Alexa fluor 488 conjugated goat anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
Calibration beads, by Bangs Laboratories (1 mentions)
Human serum albumin (hsa), by Merck Group (1 mentions)
5 protocols using dogs nta
1
MHC/Peptide-TCR Interactions by SPR
2
Liposome Preparation for Membrane Tethering
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All of the non-fluorescent lipids for liposome preparation, including POPC (1-palmitoyl-2-oleoyl-phosphatidylcholine), POPE
(1-palmitoyl-2-oleoyl-phosphatidylethanolamine), liver PI (phosphatidylinositol), POPS (1-palmitoyl-2-oleoylphosphatidylserine), cholesterol, and DOGS-NTA (1,2dioleoyl-sn-glycero-3-{[N-(5-amino-1-carboxypentyl) iminodiacetic acid]-succinyl}), were from Avanti Polar Lipids. The two fluorescence-labeled lipids, Rh-PE (rhodamine-PE) and FL-PE (fluorescein-PE), were from Molecular Probes. Lipid mixes used were prepared in chloroform with the lipid compositions of 41% (mol/mol) POPC, 17% POPE, 10% liver PI, 5% POPS, 20% cholesterol, 6% DOGS-NTA, and 1% Rh-PE or FL-PE, dried up by evaporating chloroform with a stream of nitrogen gas, and subsequently resuspended in RB150 containing 5 mM MgCl 2 and 1 mM DTT (final 8 mM lipids) by vortexing vigorously and incubating with agitation (37°C, 1 h). After freeze-thawing in liquid nitrogen and a water bath at 30°C, lipid suspensions were extruded 25 times through polycarbonate filters (pore diameters, 200 nm; Avanti Polar Lipids) in a mini-extruder (Avanti Polar Lipids) preheated at 40°C. The liposome solutions prepared were stored at 4°C Arf6-mediated membrane tethering 3 and used within a week for liposomes turbidity assays and fluorescence microscopy.
(1-palmitoyl-2-oleoyl-phosphatidylethanolamine), liver PI (phosphatidylinositol), POPS (1-palmitoyl-2-oleoylphosphatidylserine), cholesterol, and DOGS-NTA (1,2dioleoyl-sn-glycero-3-{[N-(5-amino-1-carboxypentyl) iminodiacetic acid]-succinyl}), were from Avanti Polar Lipids. The two fluorescence-labeled lipids, Rh-PE (rhodamine-PE) and FL-PE (fluorescein-PE), were from Molecular Probes. Lipid mixes used were prepared in chloroform with the lipid compositions of 41% (mol/mol) POPC, 17% POPE, 10% liver PI, 5% POPS, 20% cholesterol, 6% DOGS-NTA, and 1% Rh-PE or FL-PE, dried up by evaporating chloroform with a stream of nitrogen gas, and subsequently resuspended in RB150 containing 5 mM MgCl 2 and 1 mM DTT (final 8 mM lipids) by vortexing vigorously and incubating with agitation (37°C, 1 h). After freeze-thawing in liquid nitrogen and a water bath at 30°C, lipid suspensions were extruded 25 times through polycarbonate filters (pore diameters, 200 nm; Avanti Polar Lipids) in a mini-extruder (Avanti Polar Lipids) preheated at 40°C. The liposome solutions prepared were stored at 4°C Arf6-mediated membrane tethering 3 and used within a week for liposomes turbidity assays and fluorescence microscopy.
3
Preparation of Giant Unilamellar Vesicles
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Giant unilamellar vesicles were prepared as previously described with slight modifications (Wu et al, 2021 (link)). A polyvinyl alcohol (PVA; Alfa Aesar, 41240, average MW of 88,000–97,000) solution was prepared in deionized water (DW) at a concentration of 100 mg/ml, and 20–30 μl of the PVA solution was spread evenly on a slide glass and then dried at 50°C for 30 min to make a thin layer of PVA. A lipid mixture containing 93% (mol/mol) POPC (Avanti Polar Lipids, 850457P), 5% DOGS‐NTA (Avanti Polar Lipids, 790404P), and 2% Cy5‐PE (Avanti Polar Lipids, 810335C) was prepared in chloroform at a concentration of 1 mg/ml. A total of 20–30 μl of the lipid mixture was dropped and gently spread on PVA‐coated glass slides. Then, the chloroform was completely removed under a vacuum for at least 1 h. To prepare a chamber for GUV formation, an O‐ring was attached to the lipid‐coated slide glass, and 200 μl of GUV formation buffer containing 200 mM sucrose, 50 mM NaCl, and 20 mM Tris (pH 8.2) was used to fill the chamber. After incubation for 1 h in the dark, the GUV suspension inside the chamber was recovered. The obtained GUV suspension was stored at 4°C until use. The size was monitored by confocal microscopy.
4
Characterizing GUV-Protein Interactions
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200 μL of GUVs prepared using the droplet transfer or electroformation procedures were incubated during 5 minutes with protein, either RepA-WH1(A31V)-mCherry or mCherry. BSA at 2.5% (w/v) was included in all incubations. For immunofluorescence, they were further incubated for 20 min at room temperature with either the monoclonal antibody B3h7 (anti-RepA-WH1 pro-amyloidogenic oligomers25 (link); 1:800), or the anti-RepA-WH1 polyclonal antibody α-WH116 (link) (1:400), or an anti-His tag monoclonal antibody (Sigma; 1:200). Samples were then washed 3 times with vesicles buffer plus 0.1 M glucose. Then, GUVs were sequentially incubated for 20 min with an Alexa Fluor-488 conjugated goat anti-mouse antibody (Molecular Probes, Eugene, OR; 1:400). His6-tagged proteins were bound to GUVs prepared as described above, but with the addition of 4% Ni2+-activated DOGS-NTA (Avanti Polar Lipids).
5
Supported Lipid Bilayer Functionalization
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SLBs were prepared using liposome deposition according to previous protocols (81 ). Briefly, clean glass coverslips affixed with adhesive chambers (ibidi) were infused with DOPC vesicles supplemented with 12.5% DOGS-NTA (Avanti Polar Lipids) before washing with HEPES-buffered saline (HBS) containing 0.1% human serum albumin (HSA, Merck-Millipore). SLBs were blocked with 5% BSA and 100 µM NiSO4 before being functionalized with His-tagged proteins (anti-CD3ε UCHT1-Fab at 30 molecules/µm2 and CD58 at 200 molecules/µm2, both produced in-house). Unbound proteins and liposomes were removed with extensive HBS/HSA washing. Protein densities were calibrated using bead-supported bilayers loaded with FPs with reference to calibration beads (Bangs Laboratories).
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