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J Mol Biol. 2010 Jul 23;400(4):675-81. doi: 10.1016/j.jmb.2010.05.045. Epub 2010 May 24.

Strategy for the use of affinity grids to prepare non-His-tagged macromolecular complexes for single-particle electron microscopy.

Author information

1
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

Abstract

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.

PMID:
20562026
PMCID:
PMC2923398
DOI:
10.1016/j.jmb.2010.05.045
[Indexed for MEDLINE]
Free PMC Article

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