engleski

Distribution of myelin molecules under conditions of raft isolation : effects of different detergents

Several types of molecules are bound to the membrane via lipid anchor that is inserted into one of the phospholipid leaflets in the membrane. Glycolipids are always situated in the outer membrane leaflet, while proteins can be anchored either in the outer leaflet (via GPI– anchor), or in the inner leaflet (via prenyl, myristyl or farnesyl anchors). Immunochemical detection of these molecules in the brain is somewhat complex because of extensive myelin sheets that block access of antibodies to cell membranes and require pre-treatment with detergents. Recently, we discovered that one of these detergents used in most of immunohistochemistry protocols is causing redistribution artifacts when used in studies of lipid - anchored molecules. Since membranes have to be made porous to enable detection of axonal epitopes, we tested several detergents hoping to find one that makes pores in membrane, but does not result in significant redistribution of lipid-anchored molecules. Using different immunochemical procedures, we studied a set of molecules involved in the myelination that are either integral membrane proteins (MAG, p75NTR), or only attached to the outer leaflet of the membrane through lipid anchor (NogoR, gangliosides, OMgp). Different detergents, detergent concentrations, incubation time and temperature, were found to affect some of these molecules, leading to apparently different immunohistochemical localization. Our results clearly demonstrate that routine immunohistochemical protocol needs to be adjusted when used for the analysis of lipid-anchored molecules and that additional evidence might be needed to confirm their real physiological localization.

glycolipids; GPI-anchored proteins; detergents

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