#protein interaction

Transcript analysis

Which method is not ideal to study tissue-specific protein-protein interaction?

Tissue-specific protein-protein interactions reflect context-dependent complexes regulating differentiation and signaling. Co-immunoprecipitation preserves native interactions by antibody capture of bait protein and associated partners from lysates, suitable for tissue extracts. RNA immunoprecipitation detects RNA-protein contacts. Western blot alone measures protein abundance but not interaction. Site-directed mutagenesis artificially introduces point mutations to test functional domains in recombinant systems, typically in heterologous cell lines, rather than probing endogenous interaction l

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Protein-Protein Interaction

The interaction in FRET is:

Förster Resonance Energy Transfer involves dipole-dipole coupling between donor fluorophore in excited state and acceptor fluorophore in ground state. No photon is emitted and reabsorbed; instead energy transfers non-radiatively through long-range electromagnetic interaction. Efficiency decays with sixth power of distance, making it exquisitely sensitive between 1-10 nm. Spectral overlap between donor emission and acceptor absorption, relative dipole orientation, and distance govern transfer. This non-radiative mechanism distinguishes FRET from radiative trivial transfer and enables detection

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Protein-Protein Interaction

Which of the following methods uses phage to display protein–protein interactions?

Phage display is a combinatorial technique for studying protein interactions and affinity selection. Foreign DNA sequences are cloned into genes encoding filamentous phage coat proteins, leading to surface presentation of peptide or protein fusions. The displayed protein retains ability to bind ligands, antibodies, or receptors immobilized on solid support. After washing, bound phages are eluted and amplified in E. coli, enabling iterative enrichment. Unlike SPR, Co-IP, and FRET which measure biophysical binding, phage display directly couples genotype to phenotype for directed evolution.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Protein-Protein Interaction

If the bait and prey interact, which component is activated?

The readout of yeast two-hybrid depends on reconstitution of a functional transcription factor at the reporter locus. Bait-DNA-binding domain fusion and prey-activation domain fusion individually cannot activate transcription. Interaction bridges them, positioning the activation domain at upstream activating sequences of reporter genes such as HIS3, ADE2, or lacZ. This triggers recruitment of Mediator and RNA polymerase II, leading to measurable transcription. Reporter activation therefore serves as indirect but powerful evidence of protein-protein association inside living yeast nuclei under

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Protein-Protein Interaction

The 'prey' in yeast two-hybrid is fused to:

In yeast two-hybrid, functionality is split between two fusion proteins. Bait carrying the DNA-binding domain occupies promoter elements but lacks activation capacity. Prey consists of library proteins fused to a transcriptional activation domain that can recruit RNA polymerase II machinery and coactivators. Only upon physical binding between bait and prey does the activation domain localize to the reporter promoter, restoring a complete transcription factor. This design allows nuclear interaction to be converted into selectable reporter expression, forming basis for high-throughput binary int

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Protein-Protein Interaction

In a Yeast Two-Hybrid system, the 'bait' is fused to:

Yeast two-hybrid exploits the modular architecture of eukaryotic transcription factors, typically Gal4, which has separable DNA-binding and activation functions. Bait, the known protein, is fused to the DNA-binding domain to tether it to the upstream activating sequence of reporter genes. This fusion alone cannot activate transcription. When prey fused to activation domain interacts with bait, the two domains are brought into proximity, reconstituting a functional activator that drives transcription of auxotrophic or colorimetric reporters, enabling detection of interaction in vivo.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Hybridization

In pull-down assay, GST binds to:

Pull-down assay is an in vitro affinity capture method to demonstrate protein-protein interactions. Glutathione S-transferase fusion bait protein is expressed recombinantly and immobilized on glutathione conjugated agarose or Sepharose beads. Glutathione, a tripeptide, shows high affinity for GST active site, enabling specific immobilization. When incubated with prey lysate, interacting partners co-precipitate and are eluted for SDS-PAGE or Western blot analysis. Binding to streptavidin, nickel, or biotin represents other tagging systems like biotin-streptavidin or His-tag-Ni-NTA, not relevant

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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