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Updated: Oct 23, 2026
Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient
Published on: September 3, 2014
Lectin-induced retardation of subcellular organelles during preparative density gradient electrophoresis: selective
A Tulp1, D Verwoerd, J Neefjes
1The Netherlands Cancer Institute/ Antoni van Leeuwenhoek Huis, Division of Tumor Biology, Amsterdam.
Abstract:
Plasma membranes (PM) are difficult to separate by conventional means from other cellular compartments. Using a density gradient electrophoresis (DGE) apparatus (7 cm, x 2.2 cm), mammalian subcellular organelles were separated from a total postnuclear supernatant. The sialic acid-binding lectin wheat germ agglutinin (WGA) permitted 1.5-fold electrophoretic retardation of plasma membranes lagging far behind endoplasmic reticulum, endosomes, Golgi and lysosomes (in order of increasing electrophoretic mobility). Mobilities of the latter organelles were not affected by wheat germ agglutinin. The retarded plasma membrane was monitored by surface iodination, the presence of Ca(++)- and Na+/K(+)-ATPases and by the presence of clathrin-coated pits using Western immunoblotting. In the presence of WGA two clathrin-containing compartments were detected; in the absence of WGA three clathrin populations were seen in the electropherogram: clathrin-coated vesicles, clathrin-coated pits (on the PM) and clathrin-coated structures on the trans-Golgi network (TGN). Both in the presence and absence of WGA, plasma membrane domains of different electrophoretic mobilities were detected.
Insights
Wheat germ agglutinin (WGA) enables the separation of plasma membranes from other cellular organelles using density gradient electrophoresis. This method aids in studying plasma membrane domains and their associated proteins.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Separating plasma membranes from other cellular components is challenging using conventional methods.
- Understanding plasma membrane composition and dynamics is crucial for cellular function.
Purpose of the Study:
- To develop and validate a novel method for isolating plasma membranes from mammalian cells.
- To investigate the distinct electrophoretic properties of plasma membranes and other organelles.
Main Methods:
- Utilized a density gradient electrophoresis (DGE) apparatus for organelle separation.
- Employed wheat germ agglutinin (WGA), a sialic acid-binding lectin, to selectively retard plasma membranes.
- Monitored plasma membrane enrichment using surface iodination, ATPase activity assays, and Western immunoblotting for clathrin.
Main Results:
- Density gradient electrophoresis with WGA effectively separated plasma membranes from endoplasmic reticulum, endosomes, Golgi, and lysosomes.
- WGA binding caused a 1.5-fold electrophoretic retardation of plasma membranes without affecting other organelles.
- Distinct plasma membrane domains with varying electrophoretic mobilities were identified, both with and without WGA.
- Analysis revealed differential clathrin localization, with WGA influencing the detection of clathrin-coated structures.
Conclusions:
- Density gradient electrophoresis in conjunction with WGA provides an effective strategy for isolating and analyzing plasma membranes.
- This technique allows for the characterization of plasma membrane heterogeneity and associated protein complexes.
- The findings offer new insights into the separation and study of cellular membranes.
