Sample Preparation
All samples subjected to analytical ultracentrifugation experiments should be thoroughly dialysed against the buffer (the gel-filtration buffer is also suitable for the run), the buffer should contain at least 100 mM salt. The best choice of experimental buffer would be phosphate or Tris-buffer containing about 150 mM NaCl.
Usually we ask for 2 mg of protein (the desirable volume for the stock solution is about 1 ml) - this is enough to carry out both sedimentation velocity and sedimentation equilibrium experiments. Analytical ultracentrifugation is a non-destructive method, so you can get your sample back if you wish to. Also a generous amount of dialysis/gel-filtration buffer is needed (10-15 ml).
If you have not got enough protein, cannot use the recommended buffer or cannot dialyse the sample for some reason please come over and consult with Alex Solovyova.
We have an 8-hole rotor (AN50Ti) which can accommodate 7 dilutions of the protein for sedimentation velocity run and 21 dilutions for sedimentation equilibrium run. It is not recommended to make less than 5 dilutions for a single protein in order to keep rigorous linear extrapolation of the data points. Also the examined concentration range should cover at least one order of magnitude.
Sample Submission
A number of details about your sample/system are required before setting the run in order to choose the best possible conditions for the run. Please complete a sample submission form and return it to us. It is advisable to discuss the proposed analytical ultracentrifugation studies with us well in advance. Please remember, the more that is known about the samples, the better the analysis that can be performed
