Sample interview questions: How do you approach the study of protein-protein interactions using techniques like co-immunoprecipitation or yeast two-hybrid assays?
Sample answer:
Approaching the study of protein-protein interactions using techniques like co-immunoprecipitation or yeast two-hybrid assays:
Co-immunoprecipitation:
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Cell Lysis and Protein Extraction: Harvest cells and lyse them using appropriate buffers and detergents to extract proteins.
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Immunoprecipitation: Incubate the cell lysate with an antibody specific to the protein of interest (bait protein). This antibody is typically conjugated to magnetic beads or agarose beads.
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Bead Washing: Wash the beads extensively to remove unbound proteins and contaminants.
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Elution: Elute the immunoprecipitated proteins from the beads using appropriate buffers or conditions, such as pH changes or denaturants.
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Analysis of Interacting Proteins: Analyze the eluted proteins using techniques such as SDS-PAGE followed by Western blotting with antibodies against the potential interacting proteins, mass spectrometry for protein identification, or functional assays to assess the nature of the interaction.
Yeast Two-Hybrid Assay:
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Construction of Fusion Proteins: Create fusion proteins by fusing the bait protein and potential interacting proteins (prey proteins) to separate domains of a transcription factor.
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Transformation of Yeast: Transform yeast cells with the fusion prote… Read full answer