The orientation of membrane proteins determined in situ by immunofluorescence staining.
Structural and functional characterization of membrane proteins includes the determination of their orientation within the membrane (integral proteins), or their exposure at either the cytosolic or extracytoplasmic surface of the membrane (peripheral proteins). We have developed an easily handled immunofluorescence-based method to investigate the exposure of antigenic epitopes at either surface of the membranes in situ. We present conditions for permeabilization of p-formaldehyde-fixed cells which allow the discrimination of epitopes exposed either at the cytosolic face of membranes, within the lumen of vesicles, or at the cell surface. The potential applications of this method include (1) the use of domain-specific antibodies as a tool to study integral membrane proteins with regard to the orientation of their carboxy-terminal and amino-terminal ends or the orientation of the loops of multispanning proteins, and (2) the assignment of the epitope of monoclonal antibodies to the cytosolic or luminal domain of integral membrane proteins with the established structure.
Hinners
I
I
Institut für Biochemie und Molekulare Zellbiologie, Universität Göttingen, Göttingen, D-37073, Germany.
Moschner
J
J
Nolte
N
N
Hille-Rehfeld
A
A
D000818:Animals; D000918:Antibody Specificity; D000954:Antigens, Surface / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D002463:Cell Membrane Permeability; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry / Q000378:metabolism; D005404:Fixatives; D005455:Fluorescent Antibody Technique; D005557:Formaldehyde; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D011108:Polymers; D011817:Rabbits; D051381:Rats; D013552:Swine
D000954:Antigens, Surface; D000939:Epitopes; D005404:Fixatives; D008565:Membrane Proteins; D011108:Polymers; D005557:Formaldehyde; C003043:paraform