Immunogold localisation of P-glycoprotein in supported lipid bilayers by transmission electron microscopy and atomic force microscopy

In this study, purified P-glycoprotein molecules, a membrane drug pump resp onsible for the multidrug resistance phenomenon, were incorporated in model membranes deposited onto solid supports, according to the method described by Puu and Gustafson (1997). The insertion of proteins into planar support ed model membranes is of interest, as the films are fundamental in biosenso r applications and for the investigation of how proteins conform and aggreg ate in a lipid environment. In our investigation, two model membranes were prepared by transferring liposomes containing P-glycoprotein to different h ydrophobic supports: (a) thin amorphous carbon films; (b) Langmuir-Blodgett lipid monolayers on mica. After the labelling of P-glycoprotein with two w ell-characterised monoclonal antibodies, MM4.17 and MRK-16, samples (a) wer e observed by transmission electron microscopy (TEM) and samples (b) by ato mic force microscopy (AFM). The comparative analysis performed by TEM and AFM allowed us to demonstrate the successful insertion of P-glycoprotein in the model membranes and thei r stability under different environmental conditions (vacuum, air and water ). P-glycoprotein appeared to maintain, after purification and insertion in lipid bilayers, a good part of its conformational features as shown by the P-glycoprotein segments bearing the specific monoclonal antibody epitopes.