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Current Pharmaceutical Analysis

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ISSN (Print): 1573-4129
ISSN (Online): 1875-676X

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Research Article

Extraction and Analysis of Lipid Raft Proteins with Detergent-and Non detergent-based Method

Author(s): Yuchu Chen, Hongbei Liu, Adu-Frimpong Michael, Chenlu Gu, Lu Zhao, Sheng Tian, Xiu Li, Xia Cao and Shanshan Tong*

Volume 19, Issue 7, 2023

Published on: 24 August, 2023

Page: [540 - 547] Pages: 8

DOI: 10.2174/1573412919666230816090557

Price: $65

Abstract

Introduction: Lipid raft is found on the cell membrane and is considered a microstructure rich in cholesterol, phospholipids and target proteins that are insoluble in nonionic detergents at low temperatures.

Methods: In this study, detergent and non-detergent methods were used to extract lipid rafts from different cells. With β-cyclodextrin as the negative control group, we analyzed and compared the effects of different extraction methods on the composition of lipid rafts in Caco-2 and U251 cells using three kinds of lysate, namely detergent method 1, detergent method 2 and non-detergent method, which could be extracted and collected via sucrose density gradient centrifugation. Western blotting and immunofluorescence were utilized to determine the location of lipid rafts via the proteins Caveolin-1 and Flotillin-1, which are the characteristic proteins P-gp and TrkA in cells. The total protein in the lipid raft was quantitatively determined through the BCA (detergent compatible) kit method.

Results: The results showed that the total amount of lipid raft proteins extracted via the detergent method was more than that of the non-detergent method, while the content of β-cyclodextrin control histone that caused disruption of lipid rafts structure was the lowest.

Conclusion: The detergent method extracted more abundant lipid rafts than the non-detergent method. Detergent method 2 did not only extract more fat raft layers, but also the extracted highest total protein content, wherein it demonstrated better extraction effect with more lipid raft layers and higher expression of target protein P-gp.

Keywords: Lipid rafts extraction, western blotting, immunofluorescence, lipid raft protein characterization, KW-detergent compatible, β-cyclodextrin.

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