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 another cell. Recently, we 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 evaluated the effects of CHAPS, Saponin, deoxycholic acid, Trappsol, Tween 20, Triton X-100, Brij 96V, Brij 98, and SDS on brain tissue sections. At room temperature, all detergents (1% concentration) extracted significant amounts of both gangliosides and Thy-1. At 4C, the extraction was weaker, but Triton X-100, CHAPS, and deoxycholic acid caused significant redistribution of GD1a and Thy-1 from gray matter into the white matter. Both redistribution and extraction were significantly augmented when sections were incubated with detergents in the presence of primary antibodies. Of the nine tested detergents, none is the ideal choice. However, Brij 96V appears to be able to sufficiently reveal myelin epitopes while causing the least amount of artifacts. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.
Heffer-Lauc
Marija
M
Department of Medical Biology, University of Osijek School of Medicine, J. Huttlera 4, 31000, Osijek, Croatia.
Viljetić
Barbara
B
Vajn
Katarina
K
Schnaar
Ronald L
RL
Lauc
Gordan
G
D000818:Animals; D001921:Brain / Q000033:anatomy & histology / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D018920:Coculture Techniques; D003902:Detergents / Q000494:pharmacology*; D005677:G(M1) Ganglioside / Q000235:genetics / Q000378:metabolism; D005732:Gangliosides / Q000235:genetics / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D007150:Immunohistochemistry; D008560:Membrane Fluidity; D051379:Mice; D018345:Mice, Knockout; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013116:Spinal Cord / Q000378:metabolism; D013696:Temperature; D018800:Thy-1 Antigens / Q000378:metabolism*
D003902:Detergents; D005732:Gangliosides; D017261:Glycosylphosphatidylinositols; D011956:Receptors, Cell Surface; D018800:Thy-1 Antigens; C021298:ganglioside, GD1a; D005677:G(M1) Ganglioside