Imaging beads-retained prey assay for rapid and quantitative protein-protein interaction
Conventional Western blot based pull-down methods involve lengthy and laborious work and the results are generally not quantitative. Here, we report the imaging beads-retained prey (IBRP) assay that is rapid and quantitative in studying protein-protein interactions. In this assay, the bait is immo...
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| Main Authors: | , , |
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| Other Authors: | |
| Format: | Article |
| Language: | English |
| Published: |
2013
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| Subjects: | |
| Online Access: | https://hdl.handle.net/10356/80893 http://hdl.handle.net/10220/9876 |
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| Institution: | Nanyang Technological University |
| Language: | English |
| Summary: | Conventional Western blot based pull-down methods involve lengthy and laborious work and the results are generally not
quantitative. Here, we report the imaging beads-retained prey (IBRP) assay that is rapid and quantitative in studying
protein-protein interactions. In this assay, the bait is immobilized onto beads and the prey is fused with a fluorescence
protein. The assay takes advantage of the fluorescence of prey and directly quantifies the amount of prey binding to the
immobilized bait under a microscope. We validated the assay using previously well studied interactions and found that the
amount of prey retained on beads could have a relative linear relationship to both the inputs of bait and prey. IBRP assay
provides a universal, fast, quantitative and economical method to study protein interactions and it could be developed to
a medium- or high-throughput compatible method. With the availability of fluorescence tagged whole genome ORFs in
several organisms, we predict IBRP assay should have wide applications. |
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