The College of Life Sciences offers AUC experiments to researchers as an internal and external service. AUC uses the sedimentation characteristics of macromolecules to determine molecular weights, oligomeric states, diffusion coefficients, and self- and hetero-association properties.
Who should use AUC and when?
AUC should be used by everyone involved in preparation or utilization of pure proteins. It is essential to know what percentage of your sample exists in an active state before beginning biochemical assays, kinetic or thermodynamic studies, crystallization trials, etc. The results of an AUC experiment can be used to predict the likelihood of successfully using the protein in subsequent experiments. The technique is non-destructive and up to 100 % of the sample can be returned for further use.
hat are the sample requirements for an AUC experiment?
Sample:Purified protein should be supplied at a concentration of 1.0 mg ml-1 (equivalent to 1 O.D. absorbance at 280nm) in a volume of 1.0 mL , along with at least 20 mLs of identical buffer (buffer that the protein has been extensively dialysed or equilibrated against). From this, dilutions are made to give three samples at 0.25, 0.50 and 0.75 mg ml-1, allowing the sedimentation behaviour to be assessed over a limited concentration range.
Buffer:AUC uses absorbance and interference optical systems to monitor the sedimentation of macromolecules. Absorbance wavelengths can be tuned from 180 – 800 nm (typically 280 nm for aromatic amino acids, 230 nm for peptide backbone, 258-260 nm for nucleic acids). It is important to avoid buffer components that absorb strongly around the 280 nm region. This means DTT, glycerol, etc. can present problems. Buffers containing less than or equal to 1 mM DTT or 5 % glycerol can be tolerated but if possible should be removed. If such components cannot be removed from the buffer, the interference optical system can be used.
Also requried:The amino acid sequence of the protein is required for the calculation of partial specific volume, and an exact buffer description is required to calculate its density and viscosity.
What types of experiments can be carried out?
Sedimentation Velocity: The standard sedimentation velocity experiment involves spinning the sample at around 50 000 rpm for 16 hours. This provides a complete characterization of the oligomeric properties of the macromolecule in solution and under high centrifugal force. This is suitable for all macromolecules. Results are usually returned the day after the sample is handed over.
Sedimentation Equilibrium:Sedimentation equilibrium spins the sample at around 12 – 20 000 rpm such that the sample does not sediment to the bottom of the sample cell but instead ‘floats’ in an equilibrium position somewhere between the top and the bottom of the sample cell. SE is particularly suitable for calculating the molecular weight of detergent-associated membrane proteins as the percentage contribution from the detergent to the protein-detergent complex can be rendered invisible. SE involves repeated runs at three speeds and at more than one wavelength, therefore it can take over a week to carry out the entire experiment. Sedimentation velocity experiments are always carried out as precursors to sedimentation equilibrium experiments.
Both experiments can be carried out at temperatures from 4 – 20 0 C.
Further details of the services we provide can be found on our web site here: http://www.xtal.dundee.ac.uk/
