Equipment available: Wyatt Heleos8+ and T-rEX detectors, Shimadzu chromatography
system. Superdex 75 & 200, Superose 6 analytical columns
Equipment location: New Biochemistry 00-059
Equipment coordinator: Dr David Staunton
Equipment charge: £50/sample or £150 for 5 sample serial dilution
Sample requirements: 0.11ml of approx. 1mg/ml
Size Exclusion Chromatography - Multi-Angle Laser Scattering (SEC-MALS)
In general, when electromagnetic radiation interacts with matter, the radiation is scattered, and reemitted in all directions. Scattering gives rise to effects such as turbidity, refraction and diffraction. The intensity of scattering, measured at a fixed wavelength as a function of angle q to the incident beam is observed by sensitive detectors at different angles to the beam. This scattering is dependent on the concentration of solute and its molar mass and, for large particles, on the scattering angle and this may be used to determine the molar mass of the solute. The angular dependence for large particles (>200 kDa) may be used to estimate size (i.e. radius of gyration).
Because accurate concentration determination is vital for estimation of molar masses, the system also includes a refractive index detector - proteins have a very consistent concentration dependent effect on refractive index of solutions. Rather than measuring the sample in a batch mode, i.e. in a cuvette, the instruments are connected to a chromatography system and size exclusion column that separates the sample components and allows buffer before and after eluted peaks to be used as a very accurate reference baseline, avoiding the elaborate buffer matching required for accurate work on batch samples.
Solution molecular mass determination The light scattering system will provide accurate estimates of molar mass for homogeneous samples and will indicate where peaks are composed of mixtures either of different oligomers (mass varies across peak) or conformers (peak shape indicates multiple species but mass is the same). The software provides estimates of polydispersity.
Protein conjugate analysis. The proportion of protein to conjugate (detergent, carbohydrate, etc.) in a peak can be determined by using the UV absorption, refractive index and light scatter signals.
Size determination. For larger proteins (>200 kDa) and some other macromolecules the amount of scattered light varies with angle of detection in a way that depends on the size (strictly radius of gyration) of the molecule. If the molar mass is also determined then inferences can be drawn about the shape of the molecule - globular or rod shaped etc.
Wyatt Dawn HELEOS-II 8-angle light scattering detector and Wyatt Optilab rEX refractive index monitor linked to a Shimadzu HPLC system comprising LC-20AD pump, SIL-20A Autosampler and SPD20A UV/Vis detector
Three analytical SEC columns are available although in special cases we can use columns supplied by the users:
Superdex 200 HR10/30 column Protein MW 10 to 600 kDa
Superdex 75 HR10/30 column Protein MW 1 to 70 kDa
Superose 6 HR10/30 column Protein MW 30 kDa to 2 MDa
These columns undergo regular cleaning and are monitored for low light scatter background noise which is crucial for acceptable results.
The eluent normally goes to waste but eluted peaks may be collected manually or with a time interval fraction collector if necessary.
Samples & buffers
The autosampler system is arranged to use 100 µL injections from 110 µL sample volume though smaller volumes are possible. A guideline concentration is 1 mg/ml protein in the sample. Smaller proteins (< 10 kDa) give low light scattering signals and may need a higher concentration. Aggregates will give an unacceptable background that will affect the calculated MW so samples must be gel filtrated before running on the SEC MALS.
Users should provide their own buffers made from AR grade or better reagents in high quality water and filtered through 0.22 µm filters. We require 150mls for one sample and an additional 50mls for each subsequent sample.
Serial dilution analysis
We have noticed that SEC MALS users are reluctant to run more than a couple of samples even when a dilution series would be the best option for characterising the solution behaviour of a protein. In order to encourage the best use of the technique we are introducing a flat charge of £150 for a contiguous series of five concentrations of the same sample in the same buffer on the same column. This represents a significant reduction in costs from the current charge of £50/run. This option will be available from August. We will review the situation again at the end of the year.
Page Last Updated: 30/05/2017 by Dr D. Staunton
© 2018 Department of Biochemistry
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