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1839752 
Journal Article 
Review 
Strategy for the Use of Affinity Grids to Prepare Non-His-Tagged Macromolecular Complexes for Single-Particle Electron Microscopy 
Kelly, DF; Dukovski, D; Walz, T 
2010 
Yes 
Journal of Molecular Biology
ISSN: 0022-2836
EISSN: 1089-8638 
400 
4 
675-681 
English 
20562026 
WOS:000280652300003 
Affinity Grids are electron microscopy (EM) grids with a pre-deposited lipid monolayer containing functionalized nickel-nitrilotriacetic acid lipids. Affinity Grids can be used to prepare His-tagged proteins for single-particle EM from impure solutions or even directly from cell extracts. Here, we introduce the concept of His-tagged adaptor molecules, which eliminate the need for the target protein or complex to be His-tagged. The use of His-tagged protein A as adaptor molecule allows Affinity Grids to be used for the preparation of virtually any protein or complex provided that a specific antibody is available or can be raised against the target protein. The principle is that the Affinity Grid is coated with a specific antibody that is recruited to the grid by His-tagged protein A. The antibody-decorated Affinity Grid can then be used to isolate the target protein directly from a cell extract. We first established this approach by preparing negatively stained specimens of both native ribosomal complexes and ribosomal complexes carrying different purification tags directly from HEK-293T cell extract. We then used the His-tagged protein A/antibody strategy to isolate RNA polymerase II, still bound to native DNA, from HEK-293T cell extract, allowing us to calculate a 25-A-resolution density map by single-particle cryo-EM. 
monolayer purification; Affinity Grid; single-particle electron microscopy; ribosome; RNA polymerase II