ProteomeLab XL-I

Beckman-Coulter ProteomeLab XL-I analytical ultracentrifuge

Room 10, basement

Access to instrument only by operator or authorized persons

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)
Selectivity:	Selective – non absorbing components not visible	Non selective – sensitive for all components
Linearity:	Linear to ~ 1.5 OD	Unlimited
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:	Minutes/scan	Few seconds/scan
Windows:	Sapphire or Quartz	Sapphire
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
Volume:	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)
Volume:	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: https://sedfitsedphat.nibib.nih.gov/default.aspx

Some Buffer Properties and Their Relation to AUC Detection

AUC discussion list: SEDFIT-L@LIST.NIH.GOV https://list.nih.gov/cgi-bin/wa.exe?A0=sedfit-l