Plasma Membrane Protein Isolation: Methods and Techniques by Creative Biostructure, Slides of Biology

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protein centrifugation speed

Plasma membrane has been proven to be more than a physical barrier between the cell and the outside

environment. Being selectively permeable to ions and organic molecules, it controls the movement of substances in

and out of cells. Besides, plasma membrane is also involved in many cellular processes, such as cell adhesion, ion

conductivity and cell signaling. Studies on membrane proteins will further elucidate the important functions played

by plasma membrane.

Isolating plasma membrane is an essential step in the study of membrane protein functions. Here at Creative

Biostructure, several different approaches are utilized to efficiently isolate plasma membranes, based on the

following principles:

1. Ionic interaction

The simplest method involves the use of cationic polylysine, which can increase cell adherence by interacting with

negatively charged cells. Cells, in this case, are attached to polylysine-coated glass plates and then ruptured by

hypotonic pressure. A plasma membrane fraction remains attached to the plates after washing away the

intracellular organelles. Trypsin digestion then releases the glycoproteins from the plasma membrane, allowing

them to be further analyzed. Glycoproteins released by trypsin digestion can be readily coupled with subsequent

proteomic studies as well. This technique is therefore a relatively “clean” and scalable approach for plasma

membrane isolation.

2. Carbohydrate-binding protein

Lectin, a carbohydrate-binding protein, is also commonly used to isolate the extensively

glycosylated plasma membrane proteins. Specifically, we use Concanavalin A (ConA), a

lectin with mannose specificity, for plasma membrane isolation. In this method, ConA is

immobilized onto magnetic beads, and isolates plasma membranes from homogenized cell

lysates. The plasma membrane proteins can then be solubilized from the beads by

detergents and used for further studies.

3. Ultracentrifugation

Creative Biostructure also uses ultracentrifugation as a conventional way to isolate plasma

membrane by sucrose or iodixanol gradient methods. To isolate the plasma membrane, a low-

speed centrifugation is normally performed first to remove the nuclei before the

ultracentrifugation is applied. We also use Percoll gradient ultracentrifugation, which is proven

to be a faster method; but removal of the Percoll is usually required before further plasma

membrane studies.

4. Extraction kits

Alternatively, easy-to-use extraction kits are commercially available for the fast isolation of membrane

proteins using a bench top centrifuge, eliminating the need for ultracentrifugation. Along with the kits,

optimized purification protocols are provided to offer consistent yield and high purity (> 90%). The whole

purification procedure can be finished within 1 hour. Proteins purified using these kits can be directly

used in enzyme assays, SDS-PAGE, Western blotting and other procedures.

Creative Biostructure offers high-quality services for MemPro™ plasma membrane isolation to facilitate

the functional studies of the associated membrane proteins. Please see the list of membrane proteins

provided by Creative Biostructure.

Please feel free to contact us for a detailed quote!

References

1.Lee YC, et al. (2015) “Lectin-magnetic beads for plasma membrane isolation”. Cold Spring Harb

Protoc doi:10.1101/pdb. prot074427.

2.Chekhun VF, et al. (2013) “Alteration in lipid composition of plasma membranes of sensitive and

resistant Guerin carcinoma cells due to the action of free and liposomal form of cisplatin”. Exp

Oncol 35(3):192-197.

3.Mun JY, et al. (2013) “Efficient adhesion-based plasma membrane isolation for cell surface N-

glycan analysis”. Anal Chem 85:7462−7470.

4.Lee YC, et al. (2008) “One-step isolation of plasma membrane proteins using magnetic beads

with immobilized concanavalin A”. Protein Expr Purif 62(2):223-229.

5.Ilangumaran S, et al. (1999) “Microdomain-dependent regulation of lck and fyn protein-tyrosine

kinases in T lymphocyte plasma membranes”. Molecular Biology of the Cell 10:891–905.

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