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G. A. Zampighi, N. H. Fain, L. M. Zampighi, S. Kohan, C. Schietroma; Conical Tomography as a Novel Method for the Identification of Single Antibody Molecules in Lens Fiber Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4082.
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© ARVO (1962-2015); The Authors (2016-present)
To demonstrate the feasibility of identifying individual protein complexes in tomographic reconstructions of lens fiber cells by labeling them with antibodies, which can be identified on the basis of their size and shape, thus revealing their target protein.
We have used conical electron tomography to reconstruct: a) meshes of F-actin, decorated with monoclonal antibodies and, b) "beaded" filaments from fibers, where soluble proteins have been extracted ("ghosts"). The specimens were imaged; then the projections were aligned and reconstructed using the weighted back projection algorithm; finally they were refined using projection matching. Visualization included 3D volume rendering and segmentation methods.
Tomograms of F-actin decorated with IgGs show networks of filaments, ~6 nm in diameter, with denser particles of 6-10 nm, jutting out at variable intervals along their length. The particles are absent in reconstructions of F-actin without antibodies and some exhibit the classical tri-lobar (Y) shape, characteristic of IgGs. "Beaded" filaments were comprised of cores, 6-8 nm in diameter and beads of ~12 nm in diameter that were spaced 32-33 nm center-to-center. Antibodies against filensin recognized epitopes at the juncture between the bead and the filament’s core.
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