Fig. 1: Biotinylation, purification, and capture of native bovine Thyroglobulin (TG) on streptavidin affinity cryo-EM grids.
From: Formation of thyroid hormone revealed by a cryo-EM structure of native bovine thyroglobulin
a Elution profile from preparative gel filtration chromatography (Superose 6 Increase GL resin) showing separation of biotinylated native TG from unreacted biotinylation reagents. Unbiotinylated control is plotted in black, and samples biotinylated with 5-, 10-, and 20-fold molar excess of biotin are plotted in the indicated colors. Asterisk, unreacted biotinylation reagents. b Analysis of TG biotinylation by adsorption to streptavidin (SA) resin. Top, streptavidin-HRP dot blots of SA resin flow-through fractions, as indicated. Bottom, silver-stained TG bands from SDS-polyacrylmide gel electrophoresis analysis of SA resin flow-through fractions, as indicated. Molecular weight marker running just below TG monomer is indicated at left. N = 3. Biotinylated TG from the 10-fold molar biotin reaction was selected for cryo-EM analysis on streptavidin affinity grids. Source data for TG SDS-PAGE analysis are provided as a Source Data file. c Schematic representation of randomly biotinylated homodimeric TG complexes (monomers are distinguished by blue and orange coloration) captured on a streptavidin affinity grid (streptavidin 2D crystal; pale blue; biotinylated lipids, red; continuous carbon, black). Stars represent biotins; tails protruding from the stars represent the flexible 60-Å linker.
