Sample interview questions: How do you approach studying protein-protein recognition and binding using biophysical techniques?
Sample answer:
1. Protein Expression and Purification:
– Express the proteins of interest in a suitable expression system (e.g., E. coli, yeast, or mammalian cells) using recombinant DNA technology.
– Optimize expression conditions to obtain high yields of soluble, functional proteins.
– Purify the proteins using appropriate chromatographic techniques (e.g., affinity, ion exchange, or size-exclusion chromatography) to obtain high purity and homogeneity.
2. Protein Characterization:
– Determine the purity and homogeneity of the purified proteins using techniques such as SDS-PAGE, Western blotting, and mass spectrometry.
– Analyze the structure and integrity of the proteins using methods such as circular dichroism (CD), fluorescence spectroscopy, and differential scanning calorimetry (DSC).
3. Isothermal Titration Calorimetry (ITC):
– Use ITC to measure the thermodynamic parameters of protein-protein interactions, such as binding affinity (Kd), enthalpy change (ΔH), and entropy change (ΔS).
– ITC allows for direct observation of the binding process and provides information about the stoichiometry and energetics of the interaction.
4. Surface Plasmon Resonance (SPR):
– Employ SPR to study the kinetics and affinity of protein-protein interactions in real-time.
– SPR allows for label-free detection of binding events and provides information about the association and dissociation rates of the interaction.
5. Fluorescence Resonance Energy Transfer (FRET):
– Utilize FRET to investigate protein-protein interactions in solution or on cell surfaces.
– By labeling the proteins with appropriate fluorophores, FRET can provide information about conformational changes, stoichiometry, and proximity of the … Read full answer