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rdf:type |
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lifeskim:mentions |
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pubmed:issue |
4
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pubmed:dateCreated |
2010-7-12
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pubmed:abstractText |
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.
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pubmed:grant |
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pubmed:commentsCorrections |
http://linkedlifedata.com/resource/pubmed/commentcorrection/20562026-10600563,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20562026-10784442,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20562026-11313498,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20562026-11313499,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20562026-12176389,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20562026-14580350,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20562026-15610738,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20562026-16343539,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20562026-16762565,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20562026-16828314,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20562026-17098194,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20562026-18347330,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20562026-18400176,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20562026-18403197,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20562026-18655791,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20562026-19258014,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20562026-19489732,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20562026-20025794,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20562026-4163007,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20562026-8742743,
http://linkedlifedata.com/resource/pubmed/commentcorrection/20562026-9052786
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
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pubmed:status |
MEDLINE
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pubmed:month |
Jul
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pubmed:issn |
1089-8638
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pubmed:author |
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pubmed:copyrightInfo |
Copyright (c) 2010 Elsevier Ltd. All rights reserved.
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pubmed:issnType |
Electronic
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pubmed:day |
23
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pubmed:volume |
400
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
675-81
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pubmed:dateRevised |
2011-7-19
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pubmed:meshHeading |
pubmed-meshheading:20562026-Antibodies,
pubmed-meshheading:20562026-Cell Extracts,
pubmed-meshheading:20562026-Cell Line,
pubmed-meshheading:20562026-Humans,
pubmed-meshheading:20562026-Macromolecular Substances,
pubmed-meshheading:20562026-Microscopy, Electron,
pubmed-meshheading:20562026-Protein Binding,
pubmed-meshheading:20562026-RNA Polymerase II,
pubmed-meshheading:20562026-Ribosomes,
pubmed-meshheading:20562026-Specimen Handling
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pubmed:year |
2010
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pubmed:articleTitle |
Strategy for the use of affinity grids to prepare non-His-tagged macromolecular complexes for single-particle electron microscopy.
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pubmed:affiliation |
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
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pubmed:publicationType |
Journal Article,
Review,
Research Support, Non-U.S. Gov't,
Research Support, N.I.H., Extramural
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