SEC-SAXS: Sample Requirement
It is difficult to define minimum sample requirement for SEC-SAXS. That is depending on how the sample distributes during SEC as well as injection volume of sample, the sample size (Mw), actual sample concentration “at X-ray position” and the degree of separation by the SEC column. Advantages to use small size analytical SEC column listed here is lower sample consumption and less sample dilution during SEC, which contributes the prevention of reducing scattering signal.
In the case of BSA (a mixture containing monomer, dimer and oligomers, monomer's Mw=66.5 kDa) which is prepared from commercial powder without reducing agent, a 50 ul injection of 5 mg/ml BSA into Superdex 200 Increase PC3.2/300 column is well sufficient for obtaining reasonable data of BSA monomer. As a matter of course, even if the total amount of sample is the same, the 12.5 ul injection of 20 mg/ml will provide better sample separation and higher sample concentration at the x-ray position (sharper peak shape).
Assuming that the sample gives monodisperse and monomodal peak on BL4-2 SEC columns, the sample concentration of 5-15 mg/ml would be a good start point for reasonably large protein (e.g. >20 kDa). If sample separation is required from mixture solution, the highest sample concentration would be encouraged and the user needs to find the minimum injection volume providing reasonable scattering signal from well-separated sample of interest. Similarly, in the case of very small size sample or difficult sample which cannot be concentrated to high, the highest sample concentration is encouraged and the user could try to increase the injection volume to obtain reasonable scattering signal. Please note that the maximum injection volume for automatic data collections is currently 100 ul.
Since some samples may still show concentration-dependences (i.e. inter-particle interactions) on our SEC-SAXS system due to higher sample concentration during SEC, it is encouraged to perform a few SEC-SAXS data collections with different injection volumes and confirm them for further analysis (e.g. 50-30-10 ul injections per sample). Therefore, we encourage the user to bring more volume of sample at the beamline. That is also safe for unexpectedsample loss during the sample preparation at the beamline (pipetting, precipitation, centrifugation (mandatory when using the beamline SEC column), evaporation, etc.).
Since some samples may still show concentration-dependences (i.e. inter-particle interactions) on our SEC-SAXS system due to higher sample concentration during SEC, it is encouraged to perform a few SEC-SAXS data collections with different injection volumes and confirm them for further analysis (e.g. 50-30-10 ul injections per sample). Therefore, we encourage the user to bring more volume of sample at the beamline. That is also safe for unexpectedsample loss during the sample preparation at the beamline (pipetting, precipitation, centrifugation (mandatory when using the beamline SEC column), evaporation, etc.).
The sample vials will be provided by BL4-2 staff. We encourage bring own sample vials and its cap (Thermo Fisher Cat #: 6ESV9-04PP and 6ASC9ST1).