Glutaraldehyde Fixation of Protein Samples for Electron Microscopy

Samples were diluted to 100 μg/ml in 20 mM HEPES pH 7.4, 150 mM NaCl, 5% glycerol, 7.5 mM glutaraldehyde (Electron Microscopy Sciences, PA) and incubated for 5 minutes before quenching the glutaraldehyde by the addition of 1 M Tris (to a final concentration of 75 mM) and 5 minutes incubation. A 5-μl drop of sample was applied to a glow-discharged carbon-coated grid (Electron Microscopy Sciences, PA, CF300-Cu) for 10–15 seconds, blotted, stained with 2% uranyl formate (Electron Microscopy Sciences, PA), blotted and air-dried. Images were obtained using a Philips EM420 electron microscope at 120 kV, 82,000× magnification, and a 4.02 Å pixel size. The RELION (57 (link)) software was used for particle picking, and 2D and 3D class averaging.