Studies of interacting protein systems using multi-wavelength AUC


  • Studying multiple components simultaneously is essential, necessitating methods that can identify and characterize them concurrently.
  • Traditional single-wavelength sedimentation velocity experiments might not offer the depth required for comprehensive analysis, especially when components share similar hydrodynamic properties.


  • Introduction of MW-AUC (Multi-wavelength AUC):
  • This technique delves deeper than single wavelength experiments by utilizing both hydrodynamic and optical properties.
  • It capitalizes on the excitation spectra of fluorescent proteins, such as Ultramarine (blue), mTFP1 (green), and mPapaya (red). Although all fluorescent proteins have a common peak at 280 nm due to tryptophan and tyrosine absorbance, their excitation spectra in the visible region are significantly different.
  • This differentiation enables MW-AUC to distinguish between spectra in a multi-wavelength AUC experiment, even when the analytes have similar hydrodynamic properties.
  • Optical Deconvolution with UltraScan:
  • The process involves comparing the spectral angle θ between two distinct spectra (u and v).
  • Effective deconvolution is possible when the angle θ is greater than zero, with the larger the angle indicating more definitive separation.
  • This separation is further enhanced by selecting wavelength regions with minimal overlap


  • Mixtures of three fluorescent proteins (Ultramarine, mTFP1, and mPapaya) were sedimented and analyzed. Relative ratios of these mixed proteins could be resolved even within the margin of pipetting errors.
  • Complete resolution of proteins was achieved in all instances.
  • Unlike single-wavelength methods, MW-AUC was able to differentiate mPapaya and mTFP1 due to their distinct spectral properties, even though they had identical sedimentation coefficients.
  • This led to accurate characterization and determination of molar ratios for multiple analytes, even in the presence of pipetting errors.
  • MW-AUC also accurately determined interactions and molar ratios of associating proteins when tagged to different fluorescent proteins, all validated by molar extinction coefficients.
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Comparability Studies
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