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Advantages of a Y2H screen include: it is relatively fast and easy way to screen for - interactions
protein-protein
Y2H requires little hands-on time and technical ______
skill
Y2H is able to be scaled up by screening yeast libraries of tagged 'prey' proteins against a single '______', allowing thousands of potential interactions to be screened rapidly
bait
Limitations of Yeast TwoHybrids: There are high rates of false positives and false ______ in Y2H screens
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Interaction must occur in the ______ for the reporter gene to be activated
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Protein folding, expression, and post-translational modifications may not be the same in yeast as they are in ______, so their physical interactions may be different
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FRET in lab uses a Confocal microscope Example Example Limitations of FRET Co-______
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Co-immunoprecipitation is an extension of IP that is based on the potential of IP reactions to capture and purify the primary target (i.e., the ______)
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In Co-IP, complexes of two (or more) proteins are isolated using a procedure similar to the IP ______
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Co-IP is often used for the analysis of interactions of multiple proteins and their ______
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A protein complex is isolated by Co-IP using an antibody for one of the components in the ______
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The choice of antibody is critical for successful Co-IP. The antibody must bind to the surface of the ______
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The Y2H assay relies on the expression of a reporter gene, which is activated by the binding of a particular ______ factor.
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The query protein of interest fused with the BD is known as the ______, and the protein library fused with the AD is referred to as the Prey.
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In order to activate the reporter gene expression, a transcriptional unit must be present at the gene locus, which is only possible if Bait and Prey ______.
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Expression of a reporter gene requires the binding of a transcription factor, which normally consists of two functionally and structurally independent domains: DNA-binding (DB) and activation (AD) ______.
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In the absence of Bait-Prey interaction, the AD domain is unable to localize to the reporter gene to drive gene ______.
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However, when Bait and Prey interact, the DB domain binds to the DNA localizing the AD domain upstream of the reporter gene, leading to the expression of reporter ______.
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The yeast 2-hybrid (Y2H) assay is a well-established technique to detect protein-protein interactions. This is an extremely powerful tool for researchers and is often used alongside one or two other methods to examine the multitude of interactions that take place in cells. The assay is straightforward to perform and generates high-quality results in a short amount of time. A yeast two-hybrid (Y2H) experiment detects the physical interactions of proteins through the downstream activation of a ______ gene. How exactly this transcription is measured depends on the ______ gene.
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Despite the significant roles of PPIs in cellular functions, they have considerable potential roles as therapeutic targets. Targeted modulation of PPIs with small molecules is one of the most promising approaches with more than 50 PPIs that have successfully been targeted with small molecules. Most of these are inhibitors of PPIs, but stabilization of PPIs is also a strategy for targeted PPI modulation. Protein-protein interactions can be classified based on ______, affinity, and lifetime kD value.
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Several enzymes, carrier proteins, scaffolding proteins, and transcriptional regulatory factors carry out their functions as ______.
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Methods to study protein-protein interactions include the yeast two-hybrid system. The yeast 2-hybrid (Y2H) assay is a well-established technique to detect protein-protein interactions. This is an extremely powerful tool for researchers and is often used alongside one or two other methods to examine the multitude of interactions that take place in cells. The assay is straightforward to perform and generates high-quality results in a short amount of time. A yeast two-hybrid (Y2H) experiment detects the physical interactions of proteins through the downstream activation of a reporter gene. How exactly this transcription is measured depends on the reporter gene. But most commonly it is done by ______ selection, i.e.
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Despite the significant roles of PPIs in cellular functions, they have considerable potential roles as therapeutic targets. Targeted modulation of PPIs with small molecules is one of the most promising approaches with more than 50 PPIs that have successfully been targeted with small molecules. Most of these are inhibitors of PPIs, but stabilization of PPIs is also a strategy for targeted PPI modulation. PPI in drug development is a field that explores the therapeutic potential of modulating protein-protein interactions through the use of small molecules. This approach has been successful in targeting over 50 PPIs with small molecules, either as inhibitors or for the ______ of PPIs.
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Protein-protein interactions play significant roles in cellular functions and have potential therapeutic implications. Targeted modulation of PPIs with small molecules is a promising approach, with over 50 PPIs successfully targeted with small molecules. Most of these are inhibitors, but another strategy is the ______ of PPIs for therapeutic purposes.
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