Visualizing Recombinant and Patient Derived Poly(GA) Proteins

To examine the filamentous structure of recombinant GST-(GA)₅₀ proteins, recombinant GST or GST-(GA)₅₀ proteins were diluted to 1 µg/µl in 20 mM Tris–HCl, pH 7.5, 50 mM KCl, 10 mM MgCl₂ in a final volume of 30 µl. The samples were incubated at 30 °C for 6 h, and then diluted to 0.1 µg/µl by reaction buffer and loaded onto grids for regular EM analysis. For immuno-EM analysis, mouse monoclonal anti-GST antibody (1:20, Thermo Scientific) was used as primary antibody, and goat anti-mouse IgG conjugated with 6 nm colloidal gold particles (1:20, Jackson ImmunoResearch Laboratories) was used as the secondary antibody. To examine the filamentous structure of poly(GA) proteins in c9FTD/ALS patients, small pieces (1.5 × 1.5 × 1 mm) of cerebellar folia or hippocampus from formalin-fixed brains were dissected and processed for routine electron microscopy (EM) or post-embedding immunogold EM as previously described [34 (link)]. Rabbit polyclonal anti-poly(GA) antibody (1:50) was used as a primary antibody and goat anti-rabbit IgG conjugated with 18 nm colloidal gold particles (1:20, Jackson ImmunoResearch Laboratories) was used as the secondary antibody. Thin sections stained with uranyl acetate and lead citrate were examined with a Philips 208S electron microscope (FEI) fitted with a Gatan 831 Orius CCD camera (Gatan). Digital images were processed with Adobe Photoshop CS5 software.