Online analytical biophysical characterization device
The SAXS analysis
of mixtures may be improved by performing a size exclusion chromatography (SEC)
step immediately prior to the SAXS measurements. Moreover, the analysis of the
SAXS data is greatly facilitated if the sample is characterized biophysically
by complementary techniques. We, therefore, offer the possibility to use a
multi-detector SEC system which allows the determination of the eluate
concentration (using refractive index RI) as well as the molecular weight (from
multi-angle laser light scattering, MALLS) and hydrodynamic radius (from
dynamic light scattering, DLS). It is, therefore, possible to use this system
to characterize the oligomeric states of individual proteins, analyse the
formation/dissociation of complexes and obtain the shape factor, Rg/Rh,
where Rg is the radius of gyration obtained from SAXS and Rh
the hydrodynamic radius obtained from dynamic light scattering (DLS). The
Wyatt-MALLS-DLS system is run in parallel and directly with the SAXS
takes at least 1—3 hrs to equilibrate columns prior to the online analysis
(depending on the state of the column and whether the column arrives in 20%
ethanol, water, or already in a buffer). Overnight equilibration is encouraged.
- Buffers must be 0.1 μm filtered.
buffer needs to be made to complete all online analyses and this buffer should
be derived from a single stock. A filter device and ddH2O is
available on site at P12. Whenever a buffer is changed (even minimally, e.g.,
the addition of 1 mM DTT), the column has to be re-equilibrated in the new
buffer for at least an hour prior to the next SEC-SAXS run. For example, four
proteins in four buffers would take 10—12 hrs + set up time to complete.
equilibration and set up, a typical online SAXS-MALLS-DLS experiment takes
approximately 1.25 hr to complete.
- Protein concentrations should be in the range of 5—15 mg/ml and there should be 50—100 μl of sample. The maximum injection volume is 100 μl. A typical injection is 75 μl. Note: lower-molecular weight species (less than 30 kDa) require higher injection concentrations (up to 20 mg/ml) and injection volumes.
- The Wyatt-MALLS-DLS system is not designed to be used as a general purification step and is perhaps best described as an analytical tool to assess molecular weight, and Rh of macromolecules. Therefore injected samples should be as pure as possible as opposed to a mixture of several proteins in solution.
- The SEC-SAXS system cannot be cooled: analyses are performed at ambient temperature.
- At present there is no way to recover the sample once it has been injected into the Wyatt system: the samples will end up in the waste and be irrecoverable.
We recommend collecting sample-changer batch-mode SAXS datasets from sub-samples of all protein samples before setting up any type of SEC-SAXS to assess the sensitivity of the sample toward radiation damage. If radiation damage is observed, this may be limited via the addition of small molecules to the SEC running buffer (assuming that these chemicals do not affect the structural integrity of the sample):
- The addition of up to 3% v/v glycerol.
- The addition of 2—5 mM DTT or TCEP-HCl (for reducing environments).
- The addition of 2—5 mM sodium ascorbate (for oxidising environments).
Please inform your local contact prior to your visit if you plan to use the Wyatt-MALLS-DLS system.