Minute plasma membrane derived lipid raft isolation kit

Lipid rafts from plasma membranes were obtained using the Minute™ Plasma Membrane-Derived Lipid Raft Isolation Kit (Invent biotechnologies) following the manufacturer’s instructions. Briefly, about 30 × 10⁶ cells were plated and treated with ATS, PVS, or vehicle for 24 h or 48 h. At the end, the cells were harvested, washed with ice-cold PBS, and centrifuged. The whole pellet obtained has been incubated in ice with the first buffer (Buffer A, containing phosphatase and protease inhibitors), followed by centrifugation through a spin-column system. The pellet obtained, containing nuclei and large cell debris, has been labeled as the “non-lipid rafts” fraction. The supernatant containing plasma membrane fraction (larger PM vesicles) has been centrifuged and treated with a non-ionic detergent containing buffer, followed by isolation of detergent-resistant fraction (white/grey-colored lipid raft) by flotation centrifugation. After aqueous phase removal, the highly enriched plasma membrane-derived lipid rafts have been labeled as the “lipid rafts” fraction. Both of the fractions obtained have been lysed in RIPA buffer.
The protein content from the lipid rafts fraction and the complementary fraction (Non-lipid rafts fraction) was determined using Bradford assay. Proteins were separated with 10% SDS-PAGE and detected by Western blot analysis.

Lipid raft isolation was performed using the Minute Plasma Membrane-Derived Lipid Raft Isolation Kit (LR-042, Invent Biotechnologies). Briefly, HCT116 cells (40 × 10⁶) were harvested in T175 flasks in their regular medium. Twenty-four hours later, the medium was removed, and cells were washed with PBS and incubated in either standard CC or FMCC. After 24 h, cells were treated with or without clotrimazole (15 μM) for a further 24 h. Thereafter, cells were washed with ice cold PBS, and the assay was performed based on the manufacturer’s instructions.

Cell monolayers were washed with PBS, and lipid rafts were extracted using the Minute Plasma Membrane-Derived Lipid Raft isolation kit (Invent Biotechnologies, Eden Prairie, MN, USA). The lipid raft concentration was determined with a bicinchoninic acid protein assay kit (CWBIO, Beijing, China). The cellular proteins were separated with 10% SDS-PAGE and detected by Western blot analysis.

Cells were collected before differentiation on DD2 by trypsinization and centrifuged at 1000× g for 3 min. The supernatant was then removed; pellets were washed with PBS and centrifuged at 1000× g for 3 min. Lipid rafts were extracted from the cell pellets using a spin-column and a nonionic detergent containing buffer provided in the Minute™ Plasma Membrane-Derived Lipid Raft Isolation Kit (Invent Biotechnologies, Plymouth, MN, USA). Proteins in lipid rafts were analyzed by Western blot analysis.

First, the transfected cells were treated with the anti-GD2 antibody ch14.18/CHO (dinutuximab beta, 1000 ng/ml, Rentschler Biopharma SE, Germany), disialoganglioside GD2 (NH4 + salt) (1000 ng/ml, Matreya LLC, USA), and recombinant mouse hepatocyte growth factor (HGF, 40 ng/ml, Solarbio, China). Then, lipid rafts were extracted using the Minute Plasma Membrane-Derived Lipid Raft Isolation Kit (Invent Biotechnologies, USA) according to the manufacturer’s instructions.