partie EM cours EMBO

EMBO Practical Course 2004: EM Workshop 1 EMBO Practical Course: Structural Characterization of Macromolecular Complexes: Modern Techniques and Strategies Grenoble Outstation, May 3-8, 2004 Workshop on Electron Microscopy, Analysis and Fitting 1. Electron microscopy (negative staining of adenovirus particles) Electron microscopy (EM) can be used to obtain qualitative (purity and homogeneity) and quantitative (concentration, size and oligomeric state) information. It is a fast method (15 min.) and gives direct imaging of the protein sample (see Figure below). An electron microscope will be used for demonstrating negative-stain preparation, imaging and data collection. Students will have the opportunity to discover the ease of this method and the important information it can provide. [1] Details. A 3µl sample of virus capsids at a concentration of ~0.1 mg/ml is adsorbed to a carbon-mica interface. The carbon film with adsorbed protein is floated onto a solution of the negative stain, which contains heavy metal ions – a 1% solution of ammonium molybdate was used in the image below. The film is picked up by a copper EM grid and then air-dried. The sample is embedded in the stain, which protects it from the EM vacuum as well as providing high contrast due to the strong scattering power of the metal ions. The samples will be imaged in a JEOL 1200 EXII electron microscope at a magnification of ~30000. Conditions of low electron dose can be used to preserve ...