Related Experiment Video
Updated: Jun 21, 2026
07:33
Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction
Published on: April 26, 2011
An adhesion-based method for plasma membrane isolation: evaluating cholesterol extraction from cells and their
Ludmila Bezrukov1, Paul S Blank, Ivan V Polozov
1Program in Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Analytical Biochemistry
|July 28, 2009
Summary
This study presents a fast, 15-minute method for isolating plasma membranes using polylysine-coated glass plates. This technique reveals that methyl-beta-cyclodextrin
Area of Science:
- Cell Biology
- Biochemistry
- Membrane Biology
Background:
- Isolating pure plasma membranes is crucial for studying cell surface functions.
- Existing methods can be time-consuming or involve harsh treatments.
- Directly accessible plasma membrane preparations are needed for accurate biochemical analysis.
Purpose of the Study:
- To develop a rapid and efficient method for isolating large quantities of directly accessible plasma membrane.
- To characterize the differential extraction of cholesterol from fibroblast cells and their plasma membranes using methyl-beta-cyclodextrin.
- To investigate the temperature dependency of cholesterol extraction by methyl-beta-cyclodextrin.
Main Methods:
- Cells were adhered to polylysine-coated glass plates.
- Hypotonic lysis with ice-cold distilled water was employed for membrane isolation.
- Optimal conditions for polylysine coating and cell attachment were determined.
- Contamination by cellular organelles was assessed and found to be minimal (<5%).
Main Results:
- A novel, rapid (15-minute) method for plasma membrane isolation was established.
- Methyl-beta-cyclodextrin was found to differentially extract cholesterol from intact cells versus isolated plasma membranes.
- The differential cholesterol extraction by methyl-beta-cyclodextrin is temperature-dependent.
- Cholesterol/phospholipid ratios in intact cells do not accurately predict methyl-beta-cyclodextrin's plasma membrane extraction properties.
Conclusions:
- The developed method provides a quick and effective way to obtain pure plasma membrane preparations.
- Methyl-beta-cyclodextrin's interaction with cellular cholesterol is complex and membrane-context dependent.
- Understanding these differential extraction properties is vital for accurate lipid analysis and drug development.
Related Concept Videos
01:20Overview Of Cell Separation And Isolation
Cell separation was first achieved in 1964 by S. H. Seal, who separated large tumor cells from the smaller blood cells using filtration. Two years later, Pohl and Hawk performed experiments on how cells respond differently to a nonuniform electric field based on the cell type. Such observations were the inception of cell separation methods, which allow isolating a single cell type from a heterogeneous sample.
01:18Detergent Purification of Membrane Proteins
Detergents are used to purify the integral proteins of the membrane. The hydrophobic portion of the detergent can replace membrane phospholipids while solubilizing the membrane proteins. When detergent monomers reach a specific concentration in a solution called critical micelle concentration (CMC), they form micelles. Above CMC, the concentration of the detergent monomers remains in equilibrium with the micelle. The number of detergent monomers present in the CMC varies for each detergent, and...