Protein Biophysical Characterization Techniques

1. Spectroscopic Techniques

• Circular Dichroism (CD) spectroscopy

• UV–Visible spectroscopy

• Intrinsic fluorescence spectroscopy (Trp/Tyr)

• Extrinsic fluorescence spectroscopy (ANS, ThT, Nile Red)

• Fluorescence lifetime measurements

• Fourier Transform Infrared (FTIR) spectroscopy

• Raman spectroscopy

• Near-UV CD (tertiary structure)

2. Hydrodynamic & Size-Based Techniques

• Dynamic Light Scattering (DLS)

• Static Light Scattering (SLS)

• Analytical Ultracentrifugation (AUC)

• Size Exclusion Chromatography (SEC)

• SEC–MALS (Multi-Angle Light Scattering)

3. Structural & Conformational Analysis

• X-ray crystallography

• Cryo-Electron Microscopy (Cryo-EM)

• Nuclear Magnetic Resonance (NMR) spectroscopy

• Small-Angle X-ray Scattering (SAXS)

• Small-Angle Neutron Scattering (SANS)

4. Amyloid & Aggregation-Specific Techniques

• Thioflavin T (ThT) fluorescence assay

• Congo Red binding and birefringence

• Transmission Electron Microscopy (TEM)

• Atomic Force Microscopy (AFM)

• Turbidity (light scattering) assays

• Seeding and aggregation kinetics analysis

5. Thermodynamic & Stability Measurements

• Differential Scanning Calorimetry (DSC)

• Differential Scanning Fluorimetry (DSF / Thermofluor)

• Thermal denaturation (CD/fluorescence-based)

• Chemical denaturation (urea, GdnHCl)

6. Surface & Interaction Studies

• Isothermal Titration Calorimetry (ITC)

• Surface Plasmon Resonance (SPR)

• Bio-layer Interferometry (BLI)

• Microscale Thermophoresis (MST)

7. Redox & Chemical State Analysis

• Free thiol estimation (Ellman’s assay)

• Disulfide bond analysis

• Redox titration assays

• Oxidation–reduction switching studies

8. Electrophoretic & Complementary Methods

• SDS-PAGE

• Native PAGE

• Blue Native PAGE

• Agarose gel electrophoresis (aggregates)