The Astbury Centre for Structural Molecular Biology

Velocity runs are performed in 2-sector cells which each require at least 0.4ml of sample, and 0.4ml buffer for the reference. Equilibrium runs are usually performed in 6-sector cells which take 0.13ml of sample in three sectors, and 0.13ml of buffer in the other three. We have two rotors with a capacity of 3 cells and 7 cells respectively. It is appropriate to use a range of concentrations in an equilibrium experiment, and do 2 or 3 replicates of each sample, whereas in a velocity experiment 1 to 3 samples is the norm, though more information about association or heterogeneity is obtained by spinning 3 or 4 different concentrations of each sample.

It is recommended that the buffer be in dialysis equilibrium with the sample. This is essential in order to get meaningful results from interference scans (which allow fast data capture and measure sedimentation of total solute). A good second-best is to perform buffer exchange on a size exclusion column such as Sephadex G25 (e.g. NAP5 from Pharmacia): equilibrate the column, then collect some of the buffer flowing through, then pass your solution of macromolecule through the column.

Choice of buffer

Surface charge on a macromolecule tends to retard its sedimentation and introduce non-ideality effects. These effects can be minimised by using a buffer with an ionic strength of at least 150 mM; a simple salt should contribute most of this ionic strength. KCl is a good salt to use because the masses of its ions are quite close. An example of an effective buffer is 10mM Tris pH 7.4, 150 mM KCl. Beware of additives such as DTT which absorb in the uv region. 1 mM DTT is usually OK; 10mM might cause problems as it oxidises. Beware too of differential oxidation of such compounds in reference and sample affecting equilibrium runs lasting several days. These recommendations are only intended as a starting point. If you have already established suitable buffer conditions use those. The AUC results are then comparable to those from other experiments performed with the same system.

What is needed

•  A slight excess of sample (at least 0.5ml for velocity, 0.15ml for equilibrium), and a generous amount of buffer (e.g. 10 to 20 ml).
•  A filled-in sample submission form, including:

Your name and contact details
An account number to charge the work to (a purchase order from users outside Leeds University)
Names for all samples to be analysed
Expected masses of samples
Approximate concentrations of samples
Full details of buffers used

•  Amino acid sequence (or composition) for proteins

The more known about the samples, the better is the analysis that can be performed.

Technique	Detection	Sample concentration required	Volume per sample	Time to do experiment
Velocity	Absorbance	OD = 0.1 to 1.2 Protein concs. up to 80mg/ml or more can be measured using wavelength away from peak.	0.42 ml	2 to 6 h
	Interference	0.05 to 10 mg/ml	0.42 ml	2 to 6 h
Equilibrium	Absorbance	OD = 0.05 to 0.4	0.13 ml	1 to 6 days
	Interference	0.25 to 10 mg/ml	0.13 ml	1 to 6 days
	Note:	If using interference optics it is essential to record a baseline of the cell filled with water. At present this can only be accomplished using the 2-sector cells.