To obtain high-quality multiwavelength sedimentation velocity analytical ultracentrifugation (MW-SV-AUC) data, samples should be well purified and of a sufficient concentration to allow observation by the absorbance and/or interference optical systems. The following suggestions should be followed to help with sample preparation:
- All molecules should be gel or column purified before use
- All molecules should be dialyzed into the same buffer and that buffer should be used as the reference in the reference sector of the AUC centerpiece
- The concentrations of all molecules of interest should be determined prior to experimental set-up
- The molecules should all be stable at the selected experimental temperature for several hours, as AUC runs tend to proceed overnight to allow for complete sedimentation
Chemicals to avoid
- TCEP should always be the reducing agent of choice, as there is appreciable UV range absorbance of both DTT and -ME. This same rule holds true for all buffer components, so it is best to determine the individual absorbance contribution from each reagent. If there is one, it is best to try and substitute with one that does not have absorbance at the selected experimental wavelength.
- Glycerol should be avoided, when possible, as it significantly alters the density and viscosity of the solution; however, if it is required, keep the concentration below 5%, where the density and viscosity contributions can still be reasonably estimated by the data analysis software.
Choice of buffers
- Most standard buffers are perfectly amenable to AUC analysis (unless they absorb at the desired wavelength for the experiment). It is crucial, though, to maintain some amount of ionic strength in the solution, at least 10 – 20 mM, to allow for sufficient electrostatic shielding of surface ions. Failure to do so could result in artefactual evidence of oligomerization, due to non-specific electrostatic contacts.
Sample concentrations
- The concentration of an AUC sample is often dependent on the manner of analysis desired for the sample (i.e., titration for interaction kinetics; purity estimates; thermodynamic characterization, etc.), but generally, concentrations of all macromolecules combined are held at or below 1 mg/mL to avoid non-ideal sedimentation due to molecular crowding. This is also dictated by the absorbance of the molecule(s) of interest, as data >1 O.D. become very noisy and difficult to analyze.
AUC sample volumes
- 12 mm centerpiece: 400 μL sample; no need for reference run with the ultrascan analysis
- 3 mm centerpiece: 100 μL sample