Reagents. l-α-Lysophosphatidylcholine from egg (Egg PC), sphingomyelin from egg (Egg SM), cholesterol (Chol), and 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) (DOGS-Ni-NTA), either dissolved in chloroform or in powder form, were purchased from Avanti Polar Lipids. Recombinant human E-selectin Fc chimera protein and TRAIL protein, both with a His-tag, were purchased from R&D Systems and Enzo Life Sciences, respectively.

Preparation of empty and ETLs. Empty liposomes were made using a lipid film hydration/extrusion method. Briefly, Egg PC, Egg SM, Chol, and DOGS-NTA-Ni were mixed at a molar ratio of 45:30:18:7 in chloroform in a test tube. A lipid film was obtained after removing chloroform completely with overnight vacuum. PBS (1 ml) was added to the test tube to hydrate the lipid film and make multilamellar liposomes. After hydration with occasional vortex for >1 hour, the lipid suspension was then subject to 20 extrusion cycles through 100-nm polycarbonate membrane filters (Nuclepore; Whatman) to produce unilamellar nanoscale liposomes. For protein conjugation, E-selectin and TRAIL proteins were incubated with freshly made liposomes for 30 min at 37°C to allow protein binding via the interaction between His-tag and Ni-NTA. To remove unbound TRAIL and E-selectin, protein conjugated liposomes were purified with high-performance liquid chromatography equipped with a size exclusion chromatography column (60 cm; TSKgel G4000PW; Tosoh Bioscience). Liposome size was measured by dynamic light scattering before and after conjugation (Zetasizer Nano ZS; Malvern Instruments Ltd.)

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.