title: Resin embedded multicycle imaging (REMI): a tool to evaluate protein domains.
abstract: Protein complexes associated with cellular processes comprise a significant fraction of all biology, but our understanding of their heterogeneous organization remains inadequate, particularly for physiological densities of multiple protein species. Towards resolving this limitation, we here present a new technique based on resin-embedded multicycle imaging (REMI) of proteins in-situ. By stabilizing protein structure and antigenicity in acrylic resins, affinity labels were repeatedly applied, imaged, removed, and replaced. In principle, an arbitrarily large number of proteins of interest may be imaged on the same specimen with subsequent digital overlay. A series of novel preparative methods were developed to address the problem of imaging multiple protein species in areas of the plasma membrane or volumes of cytoplasm of individual cells. For multiplexed examination of antibody staining we used straightforward computational techniques to align sequential images, and super-resolution microscopy was used to further define membrane protein colocalization. We give one example of a fibroblast membrane with eight multiplexed proteins. A simple statistical analysis of this limited membrane proteomic dataset is sufficient to demonstrate the analytical power contributed by additional imaged proteins when studying membrane protein domains.
authors: Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA.
authors: B L
authors: BL
authors: Busse
authors: Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA.
authors: L
authors: L
authors: Bezrukov
authors: Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA.
authors: P S
authors: PS
authors: Blank
authors: Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA.
authors: J
authors: J
authors: Zimmerberg
mesh_terms: D000180:Acrylic Resins; D000906:Antibodies / Q000737:chemistry; D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure*; D005347:Fibroblasts / Q000378:metabolism / Q000648:ultrastructure*; D015870:Gene Expression; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics / Q000378:metabolism; D007091:Image Processing, Computer-Assisted / Q000379:methods; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D057054:Molecular Imaging / Q000295:instrumentation / Q000379:methods*; D011107:Polylysine; D000072417:Protein Domains; D013194:Staining and Labeling / Q000379:methods*; D016610:Tissue Embedding / Q000379:methods*; D019076:Transgenes
chemical_list: D000180:Acrylic Resins; D000906:Antibodies; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D008565:Membrane Proteins; C483468:hemagglutinin, human influenza A virus; D011107:Polylysine; C048707:LR white