Williamson, R.C. and Bass, M.D. orcid.org/0000-0002-8854-9705 (2015) Comparing the Affinity of GTPase-binding Proteins using Competition Assays. JOVE-JOURNAL OF VISUALIZED EXPERIMENTS (104). ARTN e53254.
Abstract
In this protocol we demonstrate a method for comparing the competition between GTPase-binding proteins. Such an approach is important for determining the binding capabilities of GTPases for two reasons: The fact that all interactions involve the same face of the GTPases means that binding events must be considered in the context of competitors, and the fact that the bound nucleotide must also be controlled means that conventional approaches such as immunoprecipitation are unsuitable for GTPase biochemistry. The assay relies on the use of purified proteins. Purified Rac1 immobilized on beads is used as the bait protein, and can be loaded with GDP, a non-hydrolyzable version of GTP or left nucleotide free, so that the signaling stage to be investigated can be controlled. The binding proteins to be investigated are purified from mammalian cells, to allow correct folding, by means of a GFP tag. Use of the same tag on both proteins is important because not only does it allow rapid purification and elution, but also allows detection of both competitors with the same antibody during elution. This means that the relative amounts of the two bound proteins can be determined accurately.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | Journal of Visualized Experiments www.jove.com Copyright © 2015 Creative Commons Attribution 3.0 License (https://creativecommons.org/licenses/by/3.0/) |
Keywords: | Molecular Biology; Issue 104; GTPase; Rac1; competition binding; nucleotide loading; cell signaling; Rho-family |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Science (Sheffield) > School of Biosciences (Sheffield) > Department of Biomedical Science (Sheffield) |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 24 Aug 2016 08:43 |
Last Modified: | 24 Aug 2016 08:43 |
Published Version: | http://dx.doi.org/10.3791/53254 |
Status: | Published |
Publisher: | JOURNAL OF VISUALIZED EXPERIMENTS |
Refereed: | Yes |
Identification Number: | 10.3791/53254 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:104006 |
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