Glass coverslips were cleaned with 1 M KOH for 10 min, rinsed with Milli-Q water, placed in 100% ethanol for 20 min, and plasma-cleaned for 5 min. Eight-well silicone chambers (80841; ibidi) were then attached to the cleaned coverslip. A liposome solution of 1 mg/ml with a lipid ratio of 96.5% DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), 2% DGS-NTA(Ni) [2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt)], 1% biotinyl-Cap-PE [1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt)], and 0.5% PEG5000-PE [1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (ammonium salt)] [mole percent; all available from Avanti Polar Lipids (DOPC, 850375C), (DGS-NTA(Ni), 790404C), (biotinyl-Cap-PE, 870273C), (PEG5000-PE, 880220C)] was created by vesicle extrusion, as described in detail elsewhere (44 ). The lipid solution was added to the wells at a 1:5 ratio with Milli-Q water along with 10 mM CaCl2 for 15 min and washed repeatedly with PBS, followed by 0.5 mM EDTA in Milli-Q water to remove the excess CaCl2. The well was then washed with PBS and incubated with 1 mM NiCl2 to recharge the NTA groups. Disruption of the lipid bilayer was avoided by maintaining 100 to 150 μl of PBS in the wells.
Dgs nta
Manufactured by Avanti Polar Lipids
Sourced in United States
The DGS-NTA is a lab equipment product offered by Avanti Polar Lipids. It is a synthetic lipid compound that contains a nitrilotriacetic acid (NTA) headgroup. The core function of the DGS-NTA is to facilitate the immobilization and purification of histidine-tagged proteins.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Biotinyl cap pe, by Avanti Polar Lipids (1 mentions)
Peg5000 pe, by Avanti Polar Lipids (1 mentions)
Texas red 1 2 dihexadecanoyl sn glycero 3 phosphoethanolamine, by Thermo Fisher Scientific (1 mentions)
Dspe peg biotin, by Avanti Polar Lipids (1 mentions)
8 hydroxypyrene 1 3 6 trisulfonic acid, by Thermo Fisher Scientific (1 mentions)
Hiprep 16 60 sephacryl s 300 hr column, by GE Healthcare (1 mentions)
1 2 dioleoyl sn glycero 3 phosphocholine dopc, by Avanti Polar Lipids (1 mentions)
Acetyl phosphate, by Merck Group (1 mentions)
Acetate kinase, by Merck Group (1 mentions)
Alexa fluor 488 succinimidyl ester, by Thermo Fisher Scientific (1 mentions)
Silica beads, by Bangs Laboratories (1 mentions)
Biotin pe, by Avanti Polar Lipids (1 mentions)
Minisart syringe filter, by Sartorius (1 mentions)
Nickel chloride, by Merck Group (1 mentions)
Whatman anotop, by GE Healthcare (1 mentions)
7 protocols using dgs nta
1
Lipid Bilayer Formation on Coverslips
2
Protein-Loaded Lipid Vesicles for Targeted Delivery
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NMVs were prepared as previously described 20. (link), 21. , using the following lipids obtained from Avanti Polar Lipids Inc. (AL, USA): dipalmitoyl phosphatidyl choline (DOPC, 850375P), dipalmitoyl phosphatidyl glycine (DPPG, 840455P), dipalmitoyl glycerol succinyl (DGS-NTA(Ni), 790404P), cholesterol (700000), and MPLA (699800). First, DOPC and DPPG phospholipids are suspended in the protein solution and form a lipid bilayer that makes up the inner layer. In this vesicle, the protein can only be incorporated in its hydrophilic portion. The phospholipid DGS-NTA (Ni), present only in the outer vesicle containing also DOPC and CHOL, permits that the histidine tail present in the RBD protein remains bound to the outer surface of the vesicles. The amount of unincorporated protein was measured using the BCA protein assay (Thermo Fisher Scientific Inc., MA, USA).
3
Lipid Composition for Membrane Studies
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1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3phosphoglycerol (DOPG), 1′,3′-bis[1,2-dioleoyl-sn-glycero-3-phospho]-glycerol (18:1 CL), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000 (DSPE-PEG-biotin), and 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (DGS-NTA) were from Avanti Polar Lipids. Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DHPE-TexasRed) was from Invitrogen.
4
Preparation and Characterization of NTA-Functionalized Liposomes
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Unilamellar liposomes (DOPC, 0.8% DGS-NTA, Avanti Polar Lipids) were prepared as previously described5 (link). Specifically, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (Avanti Polar Lipids) was combined with 0.8% 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) (DGS-NTA[Ni]) (Avanti Polar Lipids), dried with N2 and incubated for 1 h in a vacuum desiccator. Lipids were resuspended in 20 mM Tris pH 8, 35 mM 8-Hydroxypyrene-1,3,6-trisulfonic acid (HPTS), 50 mM p-xylene-bis-pyridinium bromide (DPX) (Thermo Fischer) and subject to 10× freeze–thaw cycles in dry ice and 42 °C water bath, and 10× extrusions using a 200 µm filter. The liposomes were then purified by gel filtration using a Hi Prep 16/60 Sephacryl S-300 HR column (GE Healthcare) in 150 mM NaCl, 20 mM Tris pH 8 buffer. Proteins were added in a ratio of 1:10,000 with liposomes, with a final liposome concentration of ~400 µM, in 150 mM citrate phosphate buffer ranging from pH 4.0 to 7.5, in 0.5 pH intervals. Assays were done in 96-well opaque plates (Corning), and fluorescence was monitored over a 20-min interval, with readings being taken every 30 s (excitation 403 nm, emission 510 nm). Data were normalized to the percentage of total HPTS fluorescence, by adding 0.3% Triton X-100.
5
Reconstitution of E. coli Polar Lipids
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E. coli polar lipid extract (EcL) and 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) DGS-NTA, from Avanti Polar Lipids, Inc. (Alabaster, AL), were kept as 10–20 g/l stocks in chloroform solutions. Alexa Fluor 488 succinimidyl ester was from Molecular Probes/Invitrogen. Silica beads (nominal diameter ∼5 μm, 10.2% suspension in DI water solution) were from Bangs Laboratories, Inc. (Fishers, IN). Acetate kinase, acetyl phosphate and GTP were from Sigma. All reactants and salts were of analytical grade, from Merck. Ethanol was spectroscopic grade, also from Merck.
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E. coli polar lipid extract (EcL) and 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) DGS-NTA, from Avanti Polar Lipids, Inc. (Alabaster, AL), were kept as 10–20 g/l stocks in chloroform solutions. Alexa Fluor 488 succinimidyl ester was from Molecular Probes/Invitrogen. Silica beads (nominal diameter ∼5 μm, 10.2% suspension in DI water solution) were from Bangs Laboratories, Inc. (Fishers, IN). Acetate kinase, acetyl phosphate and GTP were from Sigma. All reactants and salts were of analytical grade, from Merck. Ethanol was spectroscopic grade, also from Merck.
6
Preparation of Biotinylated and Histidine-Tagged Lipid Vesicles
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Vesicle solutions containing (i) 0.05% of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt) (biotin-PE, Avanti® Polar Lipids, Inc) mixed with 99.95% of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, Avanti® Polar Lipids, Inc) or (ii) 5% or 10% 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) (DGS-NTA, Avanti® Polar Lipids, Inc) mixed with 95% or 90% POPC, respectively, were prepared at a concentration of 0.5 mg/mL by the following protocol. The different lipids were first mixed in 100 µL chloroform. To remove the chloroform, the lipids were dried using a N2 gas flow for 10 minutes. After evaporation of chloroform the vesicles were suspended and thoroughly mixed in 1 mL of filtered (0.2 µm Minisart® Syringe filter, Sartorius) washing buffer: 150 mM NaCl, 10 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES, Sigma), pH 7.4 for all lipids. The vesicles were then incubated on ice for 1–2 h followed by tip sonification with a CV18 model tip sonicator (Chemical instruments AB) for 15 minutes with a pulse time of 10 s and an amplitude of 55%. The lipid stock solutions were stored at −20 °C in chloroform and the vesicle solutions at 4 °C until used.
7
Supported Lipid Bilayer Preparation
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For the preparation of supported lipid bilayers, the lipid 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) (18:1 DGS-NTA(Ni) in chloroform, 790404C, Avanti Polar Lipids Inc., USA) was aliquoted, lyophilized and then hydrated using a PBS buffer at a concentration of 20 mg/mL It was extruded through a 0.1 μm filter (Whatman Anotop, GE Healthcare, USA) to create unilamellar vesicles and stored at 4 °C. Glass-bottom Petri dishes were cleaned with 1 M NaOH (Fluka) for 40 min, and then coated with vesicle suspensions to prepare the supported bilayer (24 ). The lipid bilayer was incubated with 10 mM nickel chloride (Sigma) solution for 10 min, followed by attachment of His-PGK molecules to the surface.
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