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 responsible 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 supported model membranes is of interest, as the films are fundamental in biosensor applications and for the investigation of how proteins conform and aggregate in a lipid environment. In our investigation, two model membranes were prepared by transferring liposomes containing P-glycoprotein to different hydrophobic supports: (a) thin amorphous carbon films; (b) Langmuir-Blodgett lipid monolayers on mica. After the labelling of P-glycoprotein with two well-characterised monoclonal antibodies, MM4.17 and MRK-16, samples (a) were observed by transmission electron microscopy (TEM) and samples (b) by atomic 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 their 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.
Ruspantini
I
I
Laboratorio di Ingegneria Biomedica, Istituto Superiore di Sanità, Rome, Italy.
Diociaiuti
M
M
Ippoliti
R
R
Lendaro
E
E
Gaudiano
M C
MC
Cianfriglia
M
M
Chistolini
P
P
Arancia
G
G
Molinari
A
A
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis*; D006801:Humans; D007150:Immunohistochemistry; D008051:Lipid Bilayers / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry; D018625:Microscopy, Atomic Force; D016253:Microscopy, Immunoelectron; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D008051:Lipid Bilayers; D008081:Liposomes