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.
8 hydroxypyrene 1 3 6 trisulfonic acid
Manufactured by Thermo Fisher Scientific
8-Hydroxypyrene-1,3,6-trisulfonic acid is a fluorescent compound used as a chemical reagent in various analytical and research applications. It is soluble in water and emits a yellow-green fluorescence when excited by ultraviolet light.
Automatically generated - may contain errors
Lab products found in correlation
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)
Dgs nta, by Avanti Polar Lipids (1 mentions)
Kolliphor p188, by Merck Group (1 mentions)
Bodipy fl, by Thermo Fisher Scientific (1 mentions)
Dna aptamer, by Integrated DNA Technologies (1 mentions)
Ni nta agarose superflow resin, by Qiagen (1 mentions)
3 protocols using 8 hydroxypyrene 1 3 6 trisulfonic acid
1
Preparation and Characterization of NTA-Functionalized Liposomes
2
Giant Unilamellar Vesicle Preparation Protocol
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The inner aqueous (IA) solution of
GUVs consisted of a PBS buffer (10 mM phosphate buffer, 2.7 mM KCl,
and 137 mM NaCl) at pH 7.4, in addition to 200 mM sucrose and 15%
v/v glycerol. The outer aqueous solution additionally contained 50
mg mL–1 of Kolliphor P-188 (Sigma-Aldrich, U.K.)
as per standard OLA protocols.49 (link) Lipids
were purchased from Sigma-Aldrich in powder form and were dissolved
in 100% ethanol to a final concentration of 100 mg mL–1. The lipid/octanol phase (LO) was prepared by diluting the lipid
stock in 1-octanol to a 4–5 mg mL–1 concentration.
A lipid mixture of 3:1 ratio 1,2-dioleoyl-sn-glycero-3-phosphocholine
with 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol)
sodium salt was used to mimic the anionic charge of typical bacterial
membranes.23 (link) 16:0 Liss Rhod PE (Avanti
Lipids, 0.5 mg mL–1 in ethanol) was doped to label
the LO phase. To monitor dye leakage from GUVs, HPTS (8-hydroxypyrene-1,3,6-trisulfonic
acid, Thermo Fisher) was diluted into the IA phase (50 μM).
GUVs consisted of a PBS buffer (10 mM phosphate buffer, 2.7 mM KCl,
and 137 mM NaCl) at pH 7.4, in addition to 200 mM sucrose and 15%
v/v glycerol. The outer aqueous solution additionally contained 50
mg mL–1 of Kolliphor P-188 (Sigma-Aldrich, U.K.)
as per standard OLA protocols.49 (link) Lipids
were purchased from Sigma-Aldrich in powder form and were dissolved
in 100% ethanol to a final concentration of 100 mg mL–1. The lipid/octanol phase (LO) was prepared by diluting the lipid
stock in 1-octanol to a 4–5 mg mL–1 concentration.
A lipid mixture of 3:1 ratio 1,2-dioleoyl-sn-glycero-3-phosphocholine
with 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol)
sodium salt was used to mimic the anionic charge of typical bacterial
membranes.23 (link) 16:0 Liss Rhod PE (Avanti
Lipids, 0.5 mg mL–1 in ethanol) was doped to label
the LO phase. To monitor dye leakage from GUVs, HPTS (8-hydroxypyrene-1,3,6-trisulfonic
acid, Thermo Fisher) was diluted into the IA phase (50 μM).
3
Recombinant Protein Expression and Purification
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Plasmids for recombinant expression of His-GFP and His-SNAP were transformed into Escherichia coli Rosetta 2 cells (Novagen) for protein expression. Cultures were grown in LB at 37 °C to an OD600 of 0.4, then temperature shifted to 16 °C for 20 min and induced using 0.5 mM IPTG at an OD600 between 0.5 and 0.7. Cultures were grown overnight at 16 °C. Cells were harvested by centrifugation and resuspended in PBS. Cells were lysed with 3 cycles of sonication and freeze–thaw and clarified by centrifugation. The supernatants were incubated with Ni-NTA agarose superflow resin (Qiagen) for 1 h at 4 °C. After extensive washing, proteins were eluted in a buffer containing 150 mM NaCl, 25 mM Tris, 250 mM imidazole, pH 8, 10% glycerol, and then dialyzed overnight to remove imidazole.
DNA aptamer (GAA GAC GAG CGG CGA GTG TTA TTA CGC TTG GAA ACA ACC CC) was purchased from Integrated DNA Technologies and contained a 5′ BG and 3′ FAM fluorophore modifications. Fluorescent dyes, BODIPY-FL and 8-Hydroxypyrene-1,3,6-Trisulfonic Acid, Trisodium Salt or pyranine (HPTS) were purchased from ThermoFisher Scientific. HPLC purified doxorubicin hydrochloride was purchased from Sigma-Aldrich. BG-GLA-NHS for the functionalization of the 3′-amino aptamer was purchased from New England Biolabs.
DNA aptamer (GAA GAC GAG CGG CGA GTG TTA TTA CGC TTG GAA ACA ACC CC) was purchased from Integrated DNA Technologies and contained a 5′ BG and 3′ FAM fluorophore modifications. Fluorescent dyes, BODIPY-FL and 8-Hydroxypyrene-1,3,6-Trisulfonic Acid, Trisodium Salt or pyranine (HPTS) were purchased from ThermoFisher Scientific. HPLC purified doxorubicin hydrochloride was purchased from Sigma-Aldrich. BG-GLA-NHS for the functionalization of the 3′-amino aptamer was purchased from New England Biolabs.
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