Equipment
The analytical ultracentrifuge is
a very high speed centrifuge equipped with an optical system for observing the
sedimentation process of biomolecules in real time. The instrument has the following specifications:
- Wavelength range 190-800 nm.
- Radial scans at three wavelengths are possible during one experiment.
- Simultaneous interference scanning.
- Reliable range of mass determination 1-5000 kDa (i.e. the mass from peptides
to viruses).
- Concentration range: for absorbance scanning 0.05-2 mg/ml, for interference
scanning 0.025-30 mg/ml
- Dissociation constant range 10-3-10-8 M
At the moment we have one 8-hole rotor, 2-sector and 6-sector cells with choice of quartz and sapphire windows. The optical system in our ProteomeLab XL-I analytical ultracentrifuge (Beckman Coulter) is comprised of two types of detection:
Absorbance
The absorbance system is basically a double-beam spectrophotometer coupled with slit which scans from the top to the bottom of the cell. The advantages of this detection method are selectivity and tolerance of imperfectly matched reference buffer. The disadvantages are long scanning time (90 sec to scan one cell) and relatively noisy signal.
Interference
The Rayleigh interference system detects the distribution of solute by difference in refractive index between the sample and buffer solutions. Interference detection is very advantageous in terms of sensitivity, detection speed (10 sec to scan one cell) and excellent signal-noise ratio. It is essential that the sample and buffer have equal chemical potential (i.e. should be thoroughly dialysed) and columns of sample and buffer sectors in the cell should be of the same length. These two requirements are very strict for this type of detection.
Interference and absorbance scans can be performed at the same time in order to compromise the advantages/disadvantages of each detection system.
