High throughput flow cytometry based yeast two‐hybrid array approach for large‐scale analysis of protein‐protein interactions

Carter, Mark B.; Edwards, Bruce S.; Cai, Hong; Sklar, Larry A.

doi:10.1002/cyto.a.21144

Cited by 14 publications

(12 citation statements)

References 30 publications

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“…Finally, the reporter gene lacZ can be applied for ultimate confirmation of the interaction in a semiquantitative (169) galactosidase assay. A more recent selection approach takes advantage of the yeast enhanced green fluorescent protein (yEGFP) as a reporter to screen for interacting pairs by fluorescence-associated cell sorting (FACS) (87)(88)(89).…”

Section: Development Of the Yeast Two-hybrid Systemmentioning

confidence: 99%

Diversity in GeneticIn VivoMethods for Protein-Protein Interaction Studies: from the Yeast Two-Hybrid System to the Mammalian Split-Luciferase System

Stynen

Tournu

Tavernier

et al. 2012

Microbiol Mol Biol Rev

179

143

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SUMMARY The yeast two-hybrid system pioneered the field of in vivo protein-protein interaction methods and undisputedly gave rise to a palette of ingenious techniques that are constantly pushing further the limits of the original method. Sensitivity and selectivity have improved because of various technical tricks and experimental designs. Here we present an exhaustive overview of the genetic approaches available to study in vivo binary protein interactions, based on two-hybrid and protein fragment complementation assays. These methods have been engineered and employed successfully in microorganisms such as Saccharomyces cerevisiae and Escherichia coli , but also in higher eukaryotes. From single binary pairwise interactions to whole-genome interactome mapping, the self-reassembly concept has been employed widely. Innovative studies report the use of proteins such as ubiquitin, dihydrofolate reductase, and adenylate cyclase as reconstituted reporters. Protein fragment complementation assays have extended the possibilities in protein-protein interaction studies, with technologies that enable spatial and temporal analyses of protein complexes. In addition, one-hybrid and three-hybrid systems have broadened the types of interactions that can be studied and the findings that can be obtained. Applications of these technologies are discussed, together with the advantages and limitations of the available assays.

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Section: Development Of the Yeast Two-hybrid Systemmentioning

confidence: 99%

Diversity in GeneticIn VivoMethods for Protein-Protein Interaction Studies: from the Yeast Two-Hybrid System to the Mammalian Split-Luciferase System

Stynen

Tournu

Tavernier

et al. 2012

Microbiol Mol Biol Rev

179

143

View full text Add to dashboard Cite

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“…We demonstrate that our method is superior to the standard approach to compensation supported by commercial flow cytometry software which uses only single stained control samples. Two investigators have published critiques (2, 3) of our manuscript to which we now respond.…”

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confidence: 99%

“…The non-linearity of the fluorescent spillover effects is raised in the critique by Rajwa (3) in which he states: “…the authors did not attempt to introduce a true nonlinear model…In fact, they seem to suggest that M obtained through Eq. (6) can be used somehow in a standard linear mixture model in order to estimate α.…”

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confidence: 99%

“…The utilized five fluorochromes are: (1) FITC (CD45-FITC Beckman Coulter, Cat# PN IM0782U), (2) PE (anti-human CD45-PE BioLegend, Cat# 304008), (3) Pacific Blue anti-human CD45-Pacific Blue BioLegend, Cat#304022), (4) PE-Cy7 (anti-human CD45-PE-Cy7 BioLegend, Cat#304016), (5) APC (CD45-APC Beckman Coulter, Cat# PN IM2473U), while the characteristics of the respective five band pass filters (center wavelength/band width in nm ) are: (1) 530/30, (2) 575/30, (3) 450/50, (4) 780/60, (5) 660/20.…”

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confidence: 99%

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Authors response to correspondence about an improved compensation method

Sugár¹,

González-Lergier²,

Sealfon³

2011

Cytometry

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“…The bead-based flow cytometry technique we have presented offers a more robust approach for quantitatively assaying equilibrium binding and kinetic parameters in comparison to some of the methods commonly used to achieve the same [36, 37, 51–53]. The assays are simple to set up on account of the following realities: (1) it is easy to generate GST tagged protein, (2) tagging of interacting partners with flow cytometry-suited fluorophores does not require elaborate work, and (3) in the case of nucleotide-binding proteins, both labeled and unlabeled nucleotides can easily be obtained commercially.…”

mentioning

confidence: 99%

Quantitative Bead-Based Flow Cytometry for Assaying Rab7 GTPase Interaction with the Rab-Interacting Lysosomal Protein (RILP) Effector Protein

Agola

Sivalingam

Cimino

et al. 2015

Methods in Molecular Biology

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Rab7 facilitates vesicular transport and delivery from early endosomes to late endosomes as well as from late endosomes to lysosomes. The role of Rab7 in vesicular transport is dependent on its interactions with effector proteins, among them Rab-interacting lysosomal protein (RILP), which aids in the recruitment of active Rab7 (GTP-bound) onto dynein–dynactin motor complexes to facilitate late endosomal transport on the cytoskeleton. Here we detail a novel bead-based flow cytometry assay to measure Rab7 interaction with the Rab-interacting lysosomal protein (RILP) effector protein and demonstrate its utility for quantitative assessment and studying drug–target interactions. The specific binding of GTP-bound Rab7 to RILP is readily demonstrated and shown to be dose-dependent and saturable enabling Kd and Bmax determinations. Furthermore, binding is nearly instantaneous and temperature-dependent. In a novel application of the assay method, a competitive small molecule inhibitor of Rab7 nucleotide binding (CID 1067700 or ML282) is shown to inhibit the Rab7–RILP interaction. Thus, the assay is able to distinguish that the small molecule, rather than incurring the active conformation, instead ‘locks’ the GTPase in the inactive conformation. Together, this work demonstrates the utility of using a flow cytometry assay to quantitatively characterize protein–protein interactions involving small GTPases and which has been adapted to high-throughput screening. Further, the method provides a platform for testing small molecule effects on protein–protein interactions, which can be relevant to drug discovery and development.

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High throughput flow cytometry based yeast two‐hybrid array approach for large‐scale analysis of protein‐protein interactions

Cited by 14 publications

References 30 publications

Diversity in GeneticIn VivoMethods for Protein-Protein Interaction Studies: from the Yeast Two-Hybrid System to the Mammalian Split-Luciferase System

Diversity in GeneticIn VivoMethods for Protein-Protein Interaction Studies: from the Yeast Two-Hybrid System to the Mammalian Split-Luciferase System

Authors response to correspondence about an improved compensation method

Quantitative Bead-Based Flow Cytometry for Assaying Rab7 GTPase Interaction with the Rab-Interacting Lysosomal Protein (RILP) Effector Protein

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