Affinity filtration coupled with capillary-based affinity purification for the isolation of protein complexes

The isolation of complex macromolecular assemblies at the concentrations required for structural analysis represents a major experimental challenge. Here we present a method that combines the genetic power of site-specific recombination in order to selectively “tag” one or more components of a prote...

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Bibliographic Details
Published in:Analytical biochemistry 2013-08, Vol.439 (1), p.47-49
Main Authors: Qureshi, M.S., Sheikh, Q.I., Hill, R., Brown, P.E., Dickman, M.J., Tzokov, S.B., Rice, D.W., Gjerde, D.T., Hornby, D.P.
Format: Article
Language:English
Subjects:
Actin-Related Protein 2-3 Complex - isolation & purification
Affinity
Arp2/3
Capillary
Chromatography, Affinity - methods
complexing
filtration
Filtration - methods
microfilaments
Protein complex
Protein purification
Proteins - isolation & purification
Saccharomyces cerevisiae Proteins - isolation & purification
site-specific recombination
yeasts
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Summary:The isolation of complex macromolecular assemblies at the concentrations required for structural analysis represents a major experimental challenge. Here we present a method that combines the genetic power of site-specific recombination in order to selectively “tag” one or more components of a protein complex with affinity-based rapid filtration and a final step of capillary-based enrichment. This modified form of tandem affinity purification produces highly purified protein complexes at high concentrations in a highly efficient manner. The application of the method is demonstrated for the yeast Arp2/3 heptameric protein complex involved in mediating reorganization of the actin cytoskeleton.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2013.03.035