The production of paracrystalline two-dimensional monolayers of purified protein molecules

Abstract

The application of the negative staining-carbon film technique to a range of different high molecular weight protein molecules is described. The influence of symmetrical and asymmetrical quaternary conformation of the protein on the success as previously described fo icosahedral virus particles by Horne and his colleagues, in much the same manner as previously described for icosahedral virus particles by Horne and his colleagues, in readily producing ordered two-dimensional monolayer arrays. Nevertheless, random and ordered multi-layer regions of apoferritin are also encountered. The other proteins studied, namely, human erythrocyte cylindrin and torin, the Bordetella pertussis ‘22S antigen’ and Escherichia coli glutamine synthetase, all of which have asymmetrical quaternary conformations based on the hollow cylinder model, produce ordered arrays to a lesser extent. Factors such as the preferential orientation of the protein on the mica and carbon film surface, the inter-molecular interactions which may tend to assist or prevent ordered monolayer formation, and the protein concentration will all influence the success achieved. The problems encountered can be approached by slight variation of the technique with respect to the negative stains used, their pH and concentration, the temperature and the length of time during which the protein solution dries on to the mica surface.