engleski

Effects of detergents on the redistribution of gangliosides and GPI-anchored proteins in brain tissue sections

Gangliosides and glycosylphosphatidylinositol (GPI)-anchored proteins contain lipid tails that tether them to the outer side of the cell membrane. This mode of association with the cell membrane enables them to take part in the organization of lipid rafts, but it also permits gangliosides and GPI-anchored proteins to be actively released from one cell and inserted into the membrane of an another cell. Recently we have reported that under conditions of lipid raft isolation Triton X-100 causes significant redistribution of both gangliosides and GPI-anchored proteins. Aiming to find a less disruptive detergent we have evaluated effects of CHAPS, Saponin, deoxycholic acid, Trappsol, Tween 20, Triton X-100, Brij 96V, Brij 98, and SDS on the distribution of GM1 and GD1a gangliosides and Thy-1 (GPI-anchored protein) in brain tissue sections at room temperature and at 4º ; C. At room temperature all detergents (in 1% concentration and incubation for 2 h) extracted significant amounts of both gangliosides and Thy-1 from the membranes. At 4º ; C the extraction was weaker, but Triton X-100, CHAPS and deoxycholic acid caused significant redistribution of GD1a and Thy-1 from grey matter into the white matter. Both redistribution and extraction were significantly augmented if sections were incubated with detergents in the presence of primary antibodies. Other detergents like Trappsol and Tween 20 apparently cause less redistribution but they are also less efficient in revelation of cryptic epitopes. Neither one of the nine tested detergents is the ideal choice. However, Brij 96V appears to be able to sufficiently reveal myelin epitopes, while causing the least amount of artifacts.

gangliosides; glycosylphosphatidylinositol (GPI)-anchored proteins; immunochemical analysis; brain; detergents; lipid rafts

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