Insertion of GPI-anchored alkaline phosphatase into supported membranes: a combined AFM and fluorescence microscopy study

A new method based on combined atomic force microscopy (AFM) and fluorescence microscopy observations, is proposed to visualize the insertion of glycosylphos- phatidyl inositol (GPI) anchored alkaline phosphatase from buffer solutions into sup­ported phospholipid bilayers. The technique involves the use of 27 nm diameter fluo­rescent latex beads covalently coupled to the amine groups of proteins. Fluorescence microscopy allows the estimation of the relative protein coverage into the membrane and also introduces a height amplification for the detection of protein/bead com­plexes with the AFM. The coupling of the beads with the amine groups is not specific; this new and simple approach opens up new ways to investigate proteins into sup­ported membrane systems.