Advantages and Limitations of Yeast Two-Hybrid Screening

Protein-protein interactions can be classified based on ______

______ proteins carry out their functions as homo-oligomers

Targeted modulation of PPIs with small molecules is a promising approach in ______ development

The yeast 2-hybrid assay is a technique used to detect ______ interactions

Yeast two-hybrid experiment detects the physical interactions of proteins through the activation of a ______ gene

Most commonly, transcription in a yeast two-hybrid experiment is measured by ______ selection

The Y2H assay relies on the expression of a reporter gene (such as lacZ or GFP), which is activated by the binding of a particular ______ factor.

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.

In order to activate the reporter gene expression, a ______ unit must be present at the gene locus.

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 ______ domains.

In the absence of Bait-Prey interaction, the AD domain is unable to localize to the reporter gene to drive gene ______.

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 ______.

Advantages of Y2H: Y2H is a relatively fast and easy way to screen for ______ interactions

Y2H requires little hands-on time and technical skill, making it ______ to use

Y2H can be scaled up by screening yeast libraries of tagged “prey” proteins against a single “bait”, allowing thousands of potential interactions to be screened ______

Limitations of Yeast Two-Hybrids: There are high rates of false positives and false negatives in Y2H ______

Protein folding, expression, and post-translational modifications may not be the same in yeast as they are in humans, affecting their physical ______

Fluorescence resonance energy transfer (FRET) is distance sensitive - if the distance is less than 10 nanometers, ______ happens

Co-immunoprecipitation is an extension of IP that is based on the potential of IP reactions to capture and purify the primary target, the ______

In Co-IP, complexes of two (or more) proteins are isolated using a procedure similar to the IP ______

Co-IP is often used for the analysis of interactions of multiple proteins and their ______

The principle of Co-IP is the same as IP, except that the proteins associated with the antigen are also ______

A protein complex is isolated by Co-IP using an antibody for one of the components in the ______

The choice of antibody is critical for successful Co-IP. The antibody must bind to the surface of the ______