SDS-digested freeze-fracture replica labeling electron microscopy to study the two-dimensional distribution of integral membrane proteins and phospholipids in biomembranes: practical procedure, interpretation and application.
Recently, we have developed a quick-freezing/freeze-fracture replica labeling technique, sodium dodecyl sulfate (SDS)-digested freeze-fracture replica labeling (SDS-FRL), to study the two-dimensional distribution of cytochemical labeling on the membrane surface and the relationship of this distribution to images of freeze-fracture replicas created by platinum shadowing. In SDS-FRL, unfixed, quick-frozen cells, after freeze-fracture and platinum/carbon shadowing, are treated with SDS. The detergent dissolves unfractured areas of the cell membranes, with the release of the cytoplasmic contents. The cytoplasmic and exoplasmic membrane surfaces can be then labeled cytochemically. Integral membrane proteins, revealed as intramembrane particles by freeze-fracture replication, which are indistinguishable on a purely morphological basis, can be selectively labeled by SDS-FRL with specific antibody. In addition, this approach can be applied to examine the transmembrane phospholipid distribution in various cell and intracellular membranes. In this review, we describe the practical procedure for SDS-FRL in detail, present its application to labeling of various membrane components, and briefly discuss the possibility of a combination of SDS-FRL with atomic force microscopy.
Fujimoto
K
K
Department of Anatomy, Faculty of Medicine, Kyoto University, Japan. fujimoto@med.kyoto-u.ac.jp
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure*; D005614:Freeze Fracturing / Q000379:methods*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008565:Membrane Proteins / Q000378:metabolism / Q000648:ultrastructure*; D008854:Microscopy, Electron / Q000379:methods*; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate
D008565:Membrane Proteins; D010743:Phospholipids; D012967:Sodium Dodecyl Sulfate