Topography of Lipid Droplet-Associated Proteins: Insights from Freeze-Fracture Replica Immunogold Labeling
Figure 1
Nomenclature for describing the aspects of membranes revealed by freeze-fracture. (a) Cell samples are rapidly frozen and fractured. The freeze-fracture process splits the membrane exposing the fracture faces. The membrane comprises a lipid bilayer with intercalated proteins. The half-membrane leaflet adjacent to the extracellular space is termed the E half; that adjacent to the protoplasm is termed the P half. The term “fracture face” is reserved for the interior views of membranes exposed by freeze fracturing, while the term “surface” is used for the true, natural surfaces of the membrane. The fracture face of the P half is thus termed the P face (or PF), while that of the E half is termed the E face (or EF). The true surfaces of the membrane are correspondingly designated the P surface and the E surface (PS and ES), respectively. (b) Close-up view of the final product in FRIL as viewed in the transmission electron microscope. A platinum-carbon replica is made of the fractured specimen. The replica is treated with SDS to remove the cellular components apart from those attached directly to the replica. Proteins still attached to the replica are then immunogold-labeled. On examination in the electron microscope, the electron dense gold label is clearly visible against the replica, marking the target molecule in the plane of the membrane. The proteins embedded in the replica are detected using a primary antibody followed by a secondary antibody coupled to a colloidal gold marker.
