Analytical ultracentrifugation - Instruct-CZ

In analytical ultracentrifugation (AUC), molecules are characterized directly in solution, often under biologically relevant conditions. In contrast to many other methods, there are no complications caused by interactions with matrices or surfaces. Also, no immobilization or labeling is necessary for the analysis. Analytical ultracentrifugation is considered to be one of the most accurate methods for determination of molar mass of the molecule. Since it is a first-principle method, no calibration is required to determine the mass. AUC is a non-destructive technique which is applicable to particles with molar masses ranging from several hundreds of Da (small peptides) to hundreds of MDa (viruses).

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Analytical ultracentrifugation - Instruct-CZ

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Beckman Coulter ProteomeLab XL-I - The instrument is equipped with absorbance optics (wavelength range 190 – 800 nm) and interference optics and can be used for both sedimentation velocity and sedimentation equilibrium experiments. Analytical ultracentrifugation has a broad applicability in science including determination of sample homogeneity, oligomeric state of proteins (or molecular weight, respectively) and can be used to assess aggregation and to study biomolecular interactions of self- and hetero-association systems (determination of stoichiometry and affinity, affinity can be determined usually in the range of 104-107 M-1).

Accessories:
  • four hole An-60 Ti rotor
  • quartz and sapphire windows
  • flow-through double sector centerpiece cells for sedimentation velocity experiments
  • six-channel cells for sedimentation equilibrium experiment
  • additional cells for special purposes available

Established methodologies and provided services:
  • Sedimentation velocity - determination of sedimentation coefficient, assessing sample heterogeneity, determination of oligomeric state of proteins, detection of aggregation in the sample, study of biomolecular interactions
  • Sedimentation equilibrium - determination of molecular weight, study of biomolecular interactions
  • Data analysis

Sample requirements:
  • both a sample and a reference buffer are required – samples should be equilibrated into the experimental buffer by dialysis or size-exclusion/desalting chromatography (crucial especially for the use of interference optical system)
  • buffer (usually 10-20 mM): buffers should not absorb at a wavelength where the sample is measured (e.g. phosphate buffers work well, TRIS and HEPES are tolerable at low concentrations for 280 nm)
  • ionic strength (at least 100-200 mM NaCl, or even higher for highly charged proteins): sufficient ionic strength is needed to prevent electrostatic interactions that would affect sedimentation process
  • if possible substances generating density gradients (glycerol, sucrose, cesium chloride) should be avoided
  • if the use of reductants (DTT, β-mercaptoethanol) is necessary, they should be used at low concentrations
  • concentrations: dependent on absorbtivity, but usually not higher than 1-2 mg/ml (for proteins)
  • volumes:
    • for SV experiment usually 450 µl of both sample (optimal loading absorbance 0.5-1.0 OD for absorbance optics, optimal loading concentration >0.1 mg/ml for interference optics) and reference is required, 1 ml volume of both is preferred
    • for SE experiment: at least 95% purity of a sample, usually 150 µl of both the sample (optimal loading absorbance 0.2-0.5 OD) and the reference

These requirements depend on the nature of experiments and a particular system. Details of the experiment should be further discussed.