Large unilamellar vesicles (LUVs) with different membrane compositions including cholesterol (Sigma), cholesterol sulfate (Sigma), soybean phosphatidylcholine and egg sphingomyelin (Lipoid) were prepared as described previously11 . Lipid content was determined using thin layer chromatography. The diameters of the vesicles were measured by dynamic light scattering (Zetamaster S, Malvern Instruments). Approximately 100 ng of the purified TCR in 100 μl 0.01% Triton X-100 containing buffer was mixed with 100 μl of 2 mM prepared liposomes, and 40 μl 0.1% Triton X-100 was added. Samples were agitated for 30 min at 4 °C, and the detergent was removed by adsorption to 2–3 mg of BioBeads SM-2 (BioRad) at 4 °C overnight. To analyze TCR nanoclusters, proteoliposomes were lysed by the detergent-supplemented buffers. Blue Native PAGE was performed as previously described50 (link).
Egg sphingomyelin
Manufactured by Lipoid
Sourced in Germany
Egg sphingomyelin is a naturally occurring lipid derived from egg yolks. It is a structural component of cell membranes and plays a role in cellular signaling processes.
Automatically generated - may contain errors
Lab products found in correlation
Cholesterol sulfate, by Merck Group (2 mentions)
Soybean phosphatidylcholine, by Lipoid (2 mentions)
Zetamaster s, by Malvern Panalytical (2 mentions)
Cholesterol, by Merck Group (2 mentions)
Bio beads sm 2, by Bio-Rad (2 mentions)
Milli q system, by Merck Group (1 mentions)
Oleic acid, by Merck Group (1 mentions)
3 protocols using egg sphingomyelin
1
Reconstitution of TCR Nanoclusters
2
Synthesis and Functionalization of Lipid Nanoparticles
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SNs were prepared following a method previously reported by our group with minor modifications.21 (link) Briefly, oleic acid (5 mg, 65–88%, Merck Group, Darmstadt, Germany), egg sphingomyelin (0.5 mg, 98%, Lipoid GmbH, Ludwigshafen, Germany) and the surfactant C16/C18-COO-C9H9O3 (0.5 mg, 96%, GalChimia S.L, A Coruña, Spain) with a lipid ratio 1:0.1:0.1 w/w were dissolved in 100 µL of absolute ethanol (99.7%, Cienytech S.L., A Coruña, Spain). All the additional lipid derivatives used to functionalize SNs, such us 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid (DTPA, 0.05 mg, >99%, Avanti Polar Lipids, Alabama, Al, USA), NOTA (0.05 mg) or an oleic acid modified polyethylene glycol (PEG, 2 kDa, 0.125 mg, Nanocs, New York, NY, USA) were included in the organic phase. Then, this organic phase was injected in 1 mL of MilliQ water (Millipore Milli-Q system) under magnetic stirring using an insulin syringe (0.5 mL, 0.33 × 12 mm ICO.C.1) and nanoemulsions (SNs, DTPA-SNs, NOTA-SNs or NOTA-PEG-SNs) were spontaneously formed. To prepare SNs coated with hyaluronic acid (NOTA-HA-SNs), the organic phase containing the lipids and NOTA was injected under stirring in 1 mL of an aqueous solution of sodium hyaluronate (HA, 170 kDa, 2 mg mL−1, >95%, Bioiberica, S.A.U, Barcelona, Spain).
3
Reconstitution of TCR Nanoclusters
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