GUVs were prepared via electroformation as essentially described28 (link),29 (link). Lipid-coated ITO slides were dried under vacuum overnight. The following lipid mixtures (in mol%) were spread on two ITO slides [the fluorescent lipid Texas Red-DHPE (Life Technologies) was added for imaging purposes]: DOPC/Texas Red-DHPE: 99.75/0.25, DOPC/TR-DHPE/cholesterol: 74.75/0.25/25, 94.75/0.25/5 DOPC/TR-DHPE/DOPS, 74.75/0.25/5/20 DOPC/TR-DHPE/DOPS/cholesterol, 59.75/0.25/20/20, DOPC/TR-DHPE/DOPS/cholesterol, 94.75/0.25/5 DOPC/TR-DHPE/DOPA, 74.75/0.25/5/20 DOPC/TR-DHPE/DOPA/cholesterol, DOPC/TR-DHPE/DOPS/cholesterol, 94.75/0.25/5 DOPC/TR-DHPE/PI(4,5)P2, 74.75/0.25/5/20 DOPC/TR-DHPE/PI(4,5)P2/cholesterol, 69.75/0.25/10/20 DOPC/TR-DHPE/PI(4,5)P2/cholesterol or 69.75/0.25/10/20 DOPC/TR-DHPE/PI(3)P/cholesterol. The two ITO slides were combined with the space between filled with sucrose (10%, w/v) and incubated at RT for 1.5 h (electroformation). The GUVs were added at room temperature to DyLight488-labeled rCPn0473 (100 µg/ml) and immediately observed on a confocal fluorescence microscope (Nikon Eclipse Ti-E with A1R confocal laser scanner, 60x oil objective, NA = 1.49). Image acquisition and analysis was performed with NIS-Elements (Nikon).
Tr dhpe
Manufactured by Thermo Fisher Scientific
TR-DHPE is a lipophilic dye used as a fluorescent probe in biological research applications. It is a useful tool for monitoring membrane dynamics and lipid organization in living cells.
Automatically generated - may contain errors
Lab products found in correlation
Guanosine triphosphate gtp, by Merck Group (2 mentions)
Bodipy gdp, by Thermo Fisher Scientific (2 mentions)
Atto 647n maleimide, by Jena Biosciences (2 mentions)
Atto 488 labeled guanosine diphosphate eda gdp atto 488, by Jena Biosciences (2 mentions)
Eda gppnp atto 488, by Jena Biosciences (2 mentions)
Guanosine diphosphate gdp, by MP Biomedicals (2 mentions)
Biotinylated anti chicken igm, by Merck Group (2 mentions)
Cy5 labeled streptavidin, by Thermo Fisher Scientific (2 mentions)
Bodipy gtp, by Thermo Fisher Scientific (2 mentions)
Eclipse ti e, by Nikon (1 mentions)
Nis element, by Nikon (1 mentions)
Texas red dhpe, by Thermo Fisher Scientific (1 mentions)
A1r confocal laser scanner, by Nikon (1 mentions)
Topfluor cholesterol, by Avanti Polar Lipids (1 mentions)
Ctxb fitc, by Merck Group (1 mentions)
Sucrose, by Merck Group (1 mentions)
Chloroform, by Merck Group (1 mentions)
Cholesterol, by Avanti Polar Lipids (1 mentions)
Ctxb alexa 647, by Thermo Fisher Scientific (1 mentions)
Tetracycline hydrochloride, by Merck Group (1 mentions)
6 protocols using tr dhpe
1
Electroformation of Fluorescent GUVs
2
Lipid Membrane Composition Analysis
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DOPC, DPPC, ganglioside GM1
(ovine brain), 1-palmitoyl-2-(dipyrrometheneboron difluoride)undecanoyl-sn-glycero-3-phosphocholine
(TopFluorPC), TopFluor-cholesterol, and cholesterol were all purchased
from Avanti Polar Lipids (Alabaster, AL). Sucrose, chloroform, europium(III)
chloride hexahydrate (99.99%), tetracycline hydrochloride (cell culture
grade, >95%), and cholera toxin B subunit FITC conjugate (CTxB-FITC)
were purchased from Sigma Aldrich. MOPS was purchased from Acros Organics.
TR-DHPE, BODIPY FL C5-ganglioside GM1 (GM1-BODIPY) (Invitrogen),
cholera toxin B subunit Alexa Fluor 647 tagged (CTxB-Alexa647), and
SDS were purchased from Thermo Fisher.
(ovine brain), 1-palmitoyl-2-(dipyrrometheneboron difluoride)undecanoyl-sn-glycero-3-phosphocholine
(TopFluorPC), TopFluor-cholesterol, and cholesterol were all purchased
from Avanti Polar Lipids (Alabaster, AL). Sucrose, chloroform, europium(III)
chloride hexahydrate (99.99%), tetracycline hydrochloride (cell culture
grade, >95%), and cholera toxin B subunit FITC conjugate (CTxB-FITC)
were purchased from Sigma Aldrich. MOPS was purchased from Acros Organics.
TR-DHPE, BODIPY FL C5-ganglioside GM1 (GM1-BODIPY) (Invitrogen),
cholera toxin B subunit Alexa Fluor 647 tagged (CTxB-Alexa647), and
SDS were purchased from Thermo Fisher.
3
Lipid Reagent Preparation and Characterization
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Dioleoyl phosphatidylcholine, dioleoyl phosphatidylserine, egg SM, dipalmitoyl phosphatidylserine, GM1, NBD‐PC, and Bodipy‐Chol were purchased from Avanti Polar Lipids. TR–DHPE, AF–CTxB, and OG–DHPE were purchased from Invitrogen. Other chemicals, including NaCl, CaCl2, MgCl2, 4‐(2‐Hydroxyethyl)piperazine‐1‐ethanesulfonic acid (HEPES), hexadecane, silicone oil AR 20, methyl‐β‐cyclodextrin, and SMase from Bacillus cereus were obtained from Sigma‐Aldrich. All the lipids were dissolved in chloroform or the mixture of chloroform and methanol, and stored in a refrigerator at −20 °C. NaCl, CaCl2, MgCl2, and HEPES were dissolved in distilled water at concentrations of 100 × 10−3, 5 × 10−3, 2 × 10−3, and 10 × 10−3m , respectively, to prepare an aqueous buffer solution, followed by titration to pH 7.4 with a 0.5 m NaOH solution. The aqueous buffer solution was stored in the refrigerator at 4 °C. AF–CTxB and SMase were dissolved in the aqueous buffer at concentrations of 50 µg mL−1 and 5 U mL−1, respectively, and they were stored in the refrigerator at 4 °C.
4
Purification and Labeling of H-Ras and SOS Proteins
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H-RasC118S C181 (H-Ras construct containing residues 1–181 with a single cysteine at position C181 used for coupling to the bilayer, henceforth simply H-Ras), SOSCat cys-lite (residues 566–1049 with following mutations: C838A, C635A, C980S, E718C), SOSDPC (residues 198–1049), SOSHDPC (residues 1–1049), and SOSHDPC(R552G) (residues 1–1049 with R552G) of human SOS1 were expressed in E. Coli and purified as previously described22 (link). Lipids were purchased from Avanti (Alabaster, AL). TR-DHPE, BODIPY-GDP and BODIPY-GTP were purchased from Invitrogen (Carlsbad, CA). ATTO 647N-maleimide, ATTO 488-labeled guanosine diphosphate (EDA-GDP-ATTO 488) and EDA-GppNp-ATTO 488 (non-hydrolyzeable analog of guanosine triphosphate) were purchased from Jena Bioscience (Jena, Germany). Guanosine triphosphate (GTP) was purchased from Sigma-Aldrich (Saint Louis, MO) and guanosine diphosphate (GDP) was purchased from MP biomedicals (Santa Ana, CA). Biotinylated anti-Chicken IgM was purchased from Sigma (#SAB3700240) and Cy5 labeled streptavidin was from Life Technologies (#43–4316).
5
Purification and Labeling of H-Ras and SOS Proteins
6
Patterned Lipid Bilayer Membranes
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DiynePC and DOPC lipids
were purchased as solids from Avanti Polar Lipids. The fluorescently
tagged lipid TR-DHPE was purchased as a solid from Life Technologies.
Template patterns were prepared as described in previous publications.40 (link),41 (link) Briefly, SLBs of DiynePC were formed on glass substrates by vesicle
spreading, and then polymerization was conducted by UV irradiation
through a photomask of the desired pattern, here, a 2-D array of 100
× 100 μm boxes. Lipid vesicles comprised of the specified
ratio of TR-DHPE to DOPC lipids were generated with standard probe
sonication procedures in pure water. To form membrane corrals, a suspension
of lipid vesicles (at a concentration of ∼0.5 mg/mL total lipid)
was incubated with a template pattern for 20 min and then rinsed with
a low ionic strength buffer (purified water, adjusted to pH 7.5 using
<0.1 mM HCl).
were purchased as solids from Avanti Polar Lipids. The fluorescently
tagged lipid TR-DHPE was purchased as a solid from Life Technologies.
Template patterns were prepared as described in previous publications.40 (link),41 (link) Briefly, SLBs of DiynePC were formed on glass substrates by vesicle
spreading, and then polymerization was conducted by UV irradiation
through a photomask of the desired pattern, here, a 2-D array of 100
× 100 μm boxes. Lipid vesicles comprised of the specified
ratio of TR-DHPE to DOPC lipids were generated with standard probe
sonication procedures in pure water. To form membrane corrals, a suspension
of lipid vesicles (at a concentration of ∼0.5 mg/mL total lipid)
was incubated with a template pattern for 20 min and then rinsed with
a low ionic strength buffer (purified water, adjusted to pH 7.5 using
<0.1 mM HCl).
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