SAXS Sample Preparation
Buffer a key to successful SAXS results
SAXS is a technique of differences, therefore it is extremely important to have a well matching buffer blank. The difference between the scatter of the macromolecule and the buffer is so low, that simply making up the "equivalent" buffer is not sufficient to get accurate subtraction.
Dialysis
We strongly recommend dialyzing the sample. To retain sample volume, we have found that Hampton dialysis buttons:
(eq. 30-50 ul size) are ideal for keeping the volume constant. To remove the sample, we use a Hamilton syringe with a blunt needle to pierce the dialysis membrane and remove the sample. Only a couple ul are lost in the process. Up to 8 buttons can fit into one 50 ml tube. This buffer exchange method is ideal if the buffer contains costly additives.
SEC size-exclusion chromatography
This is another excellent method to obtain a well matching buffer blank. Wait until the end of the run when chromatogram has completely baselined before collecting your buffer aliquot.
Spin or Vacuum Concentrators
The buffer in the concentrator flow through can also be a good buffer match, but depends on your buffer components. It is important to wash the filter with at least three runs of water or buffer before loading your sample.
Salt
Salt increases the background, but we've gotten good signal with up to 1M salt. The concentration of the macromolecule has more of an impact on signal than the buffer, so if the sample is monodispersed in high salt, put it in high salt.
1-2% glycerol cuts radiation damage down quite a bit, although we have seen oligomerization induced by addition of glycerol.
If you have the quantity available, trying several differnet buffers is recommended. For a start, ideal buffers might contain 100-200 mM salt with 2% glycerol.
Monodispersity SAXS samples must be homogenous
The most common problem at the beamline is aggregation in the sample. Since larger particles scatter X-rays more strongly then small particles, aggregation will bias the results. We strongly recommend doing either DLS, native gel, or gel filtration (best). If enough sample is available, we recommend conducting an additive screen to find stabilizing conditions for your construct.
Concentration finding the sweet spot for your sample
The higher the concentration, the better the signal, However, there is a balance between problems with aggregation/oligomerisation at higher concentrations, unless the macromolecule is well-behaved. We generally recommend 1-5 mg/ml and doing a concentration series.
Ideally, data should be collected on at least three different concentrations of the macromolecule in the range of 1-10 mg/ml to identify any concentration dependent behavior. Aggregation precludes data analyses.
Radiation Damage how to reduce your sample's sensitivity to radiation
Changes in the SAXS scattering curve due to radiation damage is one of the most common issues we see. Typically, long exposures showing radiation damage can be merged with short exposures to remove the damaged region while retaining the high signal to noise at the wide angles. Glycerol is a good radical scavenger, so 2% or so is good.
Detergent difficult, but possible
We recommend to not use detergents over the entire protein preparation. We observed only a few cases (~1%) where the detergent (bellow the CMC concentration) did not affect the protein signal.