Understanding protein-protein interactions is crucial in proteomics, as these interactions drive cellular functions. Various methods, like Yeast Two-Hybrid and Co-Immunoprecipitation, help researchers identify and analyze these interactions, revealing insights into biological processes and disease mechanisms.
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Yeast Two-Hybrid (Y2H) System
- Utilizes a yeast model to detect protein-protein interactions by reconstituting a functional transcription factor.
- Involves the fusion of two proteins to separate domains of a transcription factor, leading to reporter gene activation.
- Allows for high-throughput screening of interactions in a cellular context.
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Co-Immunoprecipitation (Co-IP)
- Involves the use of antibodies to isolate a target protein along with its interacting partners from a complex mixture.
- Provides information about the presence and strength of protein interactions in native conditions.
- Can be combined with mass spectrometry for identification of co-precipitated proteins.
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Pull-Down Assays
- Uses a bait protein immobilized on a solid support to capture interacting proteins from a solution.
- Typically employs tagged proteins (e.g., GST, His-tag) for easy purification and detection.
- Useful for studying direct interactions and can be performed in vitro.
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Fluorescence Resonance Energy Transfer (FRET)
- Measures energy transfer between two fluorescently labeled proteins when in close proximity, indicating interaction.
- Provides real-time, quantitative data on protein interactions in live cells.
- Requires careful selection of donor and acceptor fluorophores for optimal sensitivity.
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Bioluminescence Resonance Energy Transfer (BRET)
- Similar to FRET but uses a bioluminescent donor, allowing for reduced background noise and improved signal-to-noise ratio.
- Enables monitoring of protein interactions in live cells without the need for external light excitation.
- Particularly useful for studying dynamic interactions in real-time.
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Surface Plasmon Resonance (SPR)
- A label-free technique that measures changes in refractive index near a sensor surface to detect binding events.
- Provides real-time kinetic data on the binding affinity and specificity of protein interactions.
- Allows for the analysis of interactions under various conditions, such as different concentrations and temperatures.
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Protein Microarrays
- High-throughput technology that allows for the simultaneous analysis of thousands of protein interactions on a single slide.
- Involves immobilizing proteins on a solid surface and probing with labeled interacting partners.
- Useful for profiling interactions across different conditions or in various biological samples.
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Proximity Ligation Assay (PLA)
- A sensitive method that detects protein interactions by using pairs of antibodies that bind to the target proteins and form a circular DNA template.
- Amplification of the DNA allows for visualization and quantification of interactions at the single-molecule level.
- Can be applied in fixed cells or tissues, providing spatial information about interactions.
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Tandem Affinity Purification (TAP)
- A two-step purification method that enhances specificity and reduces background by using two different affinity tags.
- Allows for the isolation of protein complexes in their native state, preserving interactions.
- Often combined with mass spectrometry for comprehensive identification of interacting partners.
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Cross-Linking Mass Spectrometry (XL-MS)
- Involves the use of cross-linking reagents to covalently link interacting proteins, followed by mass spectrometry analysis.
- Provides structural information about protein complexes and can identify interaction interfaces.
- Useful for studying transient interactions and mapping protein conformations in complex mixtures.