Pulldown assay

A pulldown assay is a method used in biochemistry to detect in vitro bonds between macromolecules , mostly protein-protein interactions . The method can be used to (engl. For an existing target molecule bait , bait ') one or more interacting partners (engl. Prey , Prey') to identify, or with other methods , such. B. the yeast two-hybrid system to confirm proven interactions.

principle

Pulldown assays are based on the affinity of the bait molecule to its prey molecule, which is bound to a matrix. For this purpose, the bait molecule is usually provided with a protein tag (label) by generating a fusion protein and bound to a suitable matrix via this . The matrix usually consists of cross-linked dextran or agarose . After the addition of possible interaction partners, e.g. B. in the form of a cell lysate and suitable washing steps, the interaction partners remain bound to the bait molecule and thus to the matrix and can then be eluted and analyzed. The pull-down assay is thus similar to immunoprecipitation , with the protein tag taking over the function of the antibody . Commonly used protein tags are e.g. B. hexa- histidine , streptavidin and glutathione-S-transferase tags. A variant of the pulldown assay is the Strep-Protein Interaction Experiment .

In addition to the classic pulldown assay for the detection of protein-protein interactions, similar methods have occasionally been used to investigate DNA- protein interactions, with biotin- labeled DNA samples being bound to the matrix via streptavidin and used as bait .

literature

Friedrich Lottspeich , Haralabos Zorbas: Bioanalytics . Spektrum Akademischer Verlag, Heidelberg 1998, ISBN 978-3-8274-0041-3 .
Hubert Rehm , Thomas Letzel: The Experimenter: Protein Biochemistry / Proteomics . 6th edition, Spektrum Akademischer Verlag, Heidelberg 2009, ISBN 978-3-8274-2312-2 .

Web links

Pull-down assays ( Memento from December 12, 2007 in the Internet Archive ) in the Promega Protein Interaction Guide (English; PDF file; 245 kB)
Pull-Down Assays - Described by Thermo Scientific

Individual evidence

↑ Kenneth K. Wu: Analysis of Protein-DNA Binding by Streptavidin-Agarose Pulldown . In: Methods in Molecular Biology . tape 338 , 2006, pp. 281-290 , doi : 10.1385 / 1-59745-097-9: 281 .

[1] Kenneth K. Wu: Analysis of Protein-DNA Binding by Streptavidin-Agarose Pulldown . In: Methods in Molecular Biology . tape 338 , 2006, pp. 281-290 , doi : 10.1385 / 1-59745-097-9: 281 .