Beckman-Coulter ProteomeLab XL-I analytical ultracentrifuge

Room  10, basement

Contact: Roman Szczepanowski

Access to instrument  only by operator or authorized persons

AUC ProteomeLab XL-I

The analytical ultracentrifuge (AUC), Beckman ProteomeLab XL-I is used to study hydrodynamic properties of macromolecules (proteins, nucleic acids, oligosaccharides, polymers,  nanoparticles and many more) in solution, under native condition. During the experiment variety of biophysical properties of macromolecules such as molecular weight, shape, sedimentation coefficient, diffusion coefficient, equilibrium constant and stoichiometry can be determined.  

Most important features of AUC:

  •       No standards are required
  •       Simultaneous interference and absorption measurements are possible
  •       Measurements in up to 3 selected wavelengths are possible
  •       Low concentrations can be used
  •       Small sample volumes are needed
  •       Samples can be recovered
  •       The  experiment can be run at temperature range from 4°C to 50°C

Two common methods that are used are: sedimentation velocity (SV) which provide information  about  molecular weight, sedimentation coefficient, dispersity and shape and sedimentation equilibrium (SE) most often used for determination of molecular weight and subunit stoichiometry and interaction of the components.



Absorbance optics (Abs)

Interference optics (If)


Selective – non absorbing components not visible

Non selective – sensitive for all components


Linear to ~ 1.5 OD


Buffer condition:

Avoid: large amounts of DTT, TRIS, HEPES, glycerol or sucrose  etc.

Exact match of sample and reference buffer (dialysis or gel filtration required)

Acquisition time:


Few seconds/scan


Sapphire or Quartz


Condition for sedimentation velocity (SV)

High speed, single speed, stability for min. 6h


Optimal volume:

2 channel cells

400 µl, 410 µl reference

2 channel cell

400 µl, 400- 405 µl reference

Rotor speed:

40k-60k rpm

50k-60k rpm

Optimal loading

absorbance: 0.5-1.2 OD

concentrations > 0.3 fringes, min. 0.5 mg/ml

Svan setting:

Continuous mode, 0.003 cm resolution


Condition for sedimentation equilibrium (SE)

Low speed, multiple speed,

Stability for 2-5 days




2 channel cell

180 µl sample, 190 µl reference.

(150 µl  sample for MW > 100 kDa)

2 channel cell

180 µl sample, 180 µl reference

(150 µl  sample for MW > 100 kDa)


6 channel cell

90 μl sample, 95 μl reference

6 channel cell

90 μl sample, 95 μl reference

Optimal loading

absorbance: 0.2-0.5 OD

concentration: > 0.3 fringes, min. 0.1 mg/ml

Scan setting

Step mode, 0.001 cm radial increment, 20 repetitions


 Based on Peter Schuck protocol.

Sample requirements:

  • Protein should be stable in the assay conditions and relatively pure ~95%.
  • Between 1 and 7 samples can be run in parallel.
  • To investigate concentration dependent oligomerization – 3-5  different concentration of protein are needed.
  • As a reference a buffer  from dialysis  (better) or flow-through from a column is needed.
  • Buffer: NO GLYCEROL  in the buffer (less than 5% if necessary). Avoid large amounts of DTT, β-mercaptoethanol, (use TCEP instead),  100 – 150 mM of the salt is optimal to avoid non- ideality (electrostatic interaction).
  • To measure density and viscosity ~ 10 mL of the buffer is needed.

Typical SE and SV experiments:

Sedimentation equilibrium experiments: 2 proteins (top, bottom panels), 3 different concentrations (a,b,c or d,e,f) and 2 speeds (blue and red line).

Sedimentation velocity experiment (consecutive scans)

Sedimentation velocity experiments: oligomerization (top panel) , interaction of the protein (green) with ligand (orange) and formation of the complex (blue).

Used software: SEDFIT and SEDPHAT (created by P. Schuck, NIH, Bethesda, USA), SEDNTERP (D. Hayes, T. Laue, J. Philo, USA), GUSSI (C. Brautigam, UT Southwestern, USA


Further reading:

Peter Schuck Web page:

Some Buffer Properties and Their Relation to AUC Detection


AUC discussion list:   SEDFIT-L@LIST.NIH.GOV