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The Dihydrofolate Reductase Protein-Fragment Complementation Assay: A Survival-Selection Assay for Large-Scale Analysis of Protein–Protein Interactions

Combining the Dihydrofolate Reductase Protein-Fragment Complementation Assay with Gene Deletions to Establish Genotype-to-Phenotype Maps of Protein Complexes and Interaction Networks.

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Protein-Fragment Complementation Assays for Large-Scale Analysis, Functional Dissection, and Spatiotemporal Dynamic Studies of Protein-Protein Interactions in Living Cells.

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Real-Time Protein-Fragment Complementation Assays for Studying Temporal, Spatial, and Spatiotemporal Dynamics of Protein-Protein Interactions in Living Cells.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Malleshaiah, Mohan Tchekanda, Emmanuelle Michnick, Stephen W.
Description	Country affiliation: Canada Author Affiliation: Malleshaiah M ( Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, Québec H3C 3J7, Canada.); Tchekanda E ( Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115.); Michnick SW ( Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, Québec H3C 3J7, Canada.)
Abstract	Here, we present detailed protocols for direct, real-time protein-fragment complementation assays (PCAs) for studying the spatiotemporal dynamics of protein-protein interactions (PPIs). The assays require the use of two fluorescent reporter proteins-the 'Venus' version of yellow fluorescent protein (vYFP), and the monomeric infrared fluorescent protein 1.4 (IFP 1.4)-or two luciferase reporter proteins-Renilla (Rluc) and Gaussia (Gluc). The luciferase PCAs can be used to study the temporal dynamics of PPIs in any cellular compartment and on membranes. The full reversibility of these PCAs assures accurate measurements of the kinetics of PPI assembly/disassembly for processes that occur anywhere in a living cell and over time frames of seconds to hours. vYFP PCA, and all PCAs based on green fluorescent protein and its variants, are irreversible and can be used to trap and visualize rare and transient complexes and follow dynamic relocalization of constitutive complexes. vYFP PCA is limited in that accurate measurements of temporal changes in PPIs are not possible owing to the slow maturation time of vYFP (minutes to hours) and the irreversibility of its PCA that traps the complexes, thereby preventing the dissociation of PPIs that, in some instances, might cause spurious mislocalization of protein complexes. The limitations of vYFP PCA are overcome with IFP PCA, which is fully reversible and thus can be used to study spatiotemporal dynamics of PPIs on the timescale of seconds. All of these PCAs are sensitive enough to detect interactions among proteins expressed at endogenous levels in vivo.
File Format	HTM / HTML
ISSN	19403402
Issue Number	11
Journal	Cold Spring Harbor Protocols
Volume Number	2016
e-ISSN	15596095
Language	English
Publisher	Cold Spring Harbor Laboratory Press
Publisher Date	2016-11-01
Publisher Place	United States
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Discipline Clinical Laboratory Techniques
Content Type	Text
Resource Type	Article
Subject	Biochemistry, Genetics and Molecular Biology

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1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
2	Indian Institute of Technology Kharagpur	Host Institute of the Project: The host institute of the project is responsible for providing infrastructure support and hosting the project	https://www.iitkgp.ac.in
3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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6	Contents and Copyright Issues	Queries related to content curation and copyright issues	content@ndl.gov.in
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Protein-Fragment Complementation Assays for Large-Scale Analysis, Functional Dissection, and Spatiotemporal Dynamic Studies of Protein-Protein Interactions in Living Cells.

Dissecting the Contingent Interactions of Protein Complexes with the Optimized Yeast Cytosine Deaminase Protein-Fragment Complementation Assay.

The Dihydrofolate Reductase Protein-Fragment Complementation Assay: A Survival-Selection Assay for Large-Scale Analysis of Protein–Protein Interactions

Protein complementation as tool for studying protein-protein interactions in living cells

Protein-fragment complementation assays for large-scale analysis of protein-protein interactions

In Planta Visualization of Protein Interactions Using Bimolecular Fluorescence Complementation (BiFC).

An infrared reporter to detect spatiotemporal dynamics of protein-protein interactions.

Assays with protein arrays.

Combining the Dihydrofolate Reductase Protein-Fragment Complementation Assay with Gene Deletions to Establish Genotype-to-Phenotype Maps of Protein Complexes and Interaction Networks.

Real-Time Protein-Fragment Complementation Assays for Studying Temporal, Spatial, and Spatiotemporal Dynamics of Protein-Protein Interactions in Living Cells.

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Protein-Fragment Complementation Assays for Large-Scale Analysis, Functional Dissection, and Spatiotemporal Dynamic Studies of Protein-Protein Interactions in Living Cells.

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Real-Time Protein-Fragment Complementation Assays for Studying Temporal, Spatial, and Spatiotemporal Dynamics of Protein-Protein Interactions in Living Cells.