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How much sample is required?
The short answer is "it depends on the sample". Although it is not possible to give a 'one size fits all' concentration, some guidelines are provided here to help determine what may be appropriate for a particular sample. For all samples, the concentration required is a balance of achieving a sufficiently strong signal while also avoiding other complications, such as interparticle interactions at higher concentrations. Due to their weak scattering contrast, protein samples require careful consideration of the appropriate sample concentration, not least because sample volumes may be limited.
For many protein samples, it is preferable to use as dilute a sample as possible due to constraints on the amount of sample available. Excessively high concentrations are also likely to suffer aggregation problems and are more likely to suffer beam damage. Data collection has been possible from sample concentrations as low as 0.01 mg/mL, but there is no single concentration that is ideal for every sample.
Beamline staff may be contacted for detailed guidance. Some considerations are outlined below:
Batch or SEC-Coflow?
The amount of protein is dependent on the system used during data collection. Typically, 3-5 mg/mL is an approximate upper limit for most samples in Batch mode and 5 - 10 mg/mL for SEC-coflow (assuming a small SEC column and a sample not too polydisperse).
Sample molecular weight?
Keep in mind that smaller molecules scatter less requiring higher concentrations.
What is the scattering contrast?
If the electron density of the solvent or buffer is high it will reduce the scattering contrast, so higher sample concentrations are required. Conversely, some samples, e.g. RNA, have a relatively high electron density, increasing scattering contrast so lower concentrations can be used in those cases.
What information is being sought?
If only Molecular Weight and oligomerisation states are desired, quite low concentrations may be used. However, if 3-D shape reconstruction is desired, good data is required in the weak high-Q-q region which necessitates higher concentrations
Is aggregation or radiation damage likely?
Proteins vary considerably in their susceptibility to aggregation and radiation damage. Check each case and consider using SEC-coflow setup for sample analysis.
Non-Protein in Solution Samples
For most non-protein samples, the SAXS beamline is typically able to collect useful data at very low concentrations. The exact concentration depends very much on the system to be investigated, it is recommended to contact beamline staff if uncertain. Some considerations are detailed below:
How much sample is available?
Low concentrations or small volumes (as low as 50 microlitres) can be used if necessary.
How strongly does the sample scatter?
A high X-ray contrast allows for very low concentrations to be used.
Is Form Factor scattering or Structure Factor scattering desired?
Structure Factor scattering, due to interparticle interactions, is increasingly significant as concentration increases. Low concentrations must be used if only Form Factor scattering is desired.
Preparing your sample
Preparing high quality protein samples for SAXS analysis usually involves quite a lot of effort. The quality of the data collected depends greatly on the quality of samples examined. The SAXS measurement itself can been thought of as the last part of a process, most of which is done before arrival at the synchrotron.
The most important considerations for protein samples are:
Is the sample monodisperse?
A highly monodisperse sample is required for the best data quality and it is recommended to check samples prior to arriving at the synchrotron. If monodispersity is only achieve during SEC use the SEC-coflow setup.
Is the sample stable?
Check if sample handles freezing or storage. If it can’t handle storage prepare it fresh before experiment or a analyse your sample through SEC-coflow to remove partial aggregation. Unstable proteins won’t benefit from SEC.
Is the buffer fully matched to the sample?
The best way to be sure is to dialyse you sample with its buffer, assuming it's stable long enough to perform dialysis. If you use SEC-coflow (without buffer radiation damage) buffer matching is ensured (ensure column is equilibrated!)
Key steps for sample preparation:
Centrifuging - all samples should be centrifuged before being loaded to the plates to remove/reduce aggregation or other contamination. Plates containing the samples should also be centrifuged to remove air bubbles. Chemistry laboratory at the synchrotron has a temperature controlled centrifuge with rotors for 1.5 mL Eppendorf tubes, large Falcon tubes and 96-well plates. A miniature centrifuge is also available at the beamline for 1.5 mL Eppendorf tubes.
Degassing - all samples should be degassed prior being loaded on the plate for SAXS analysis. In the case of the batch setup presence of bubble with damage your data while in the case of SEC-coflow with can also cause problems in the column and equipment. If using SEC-coflow setup ensure that buffers and water used in the SEC system has been filtered and degassed.
Protocol recommended
Centrifuge sample in Eppendorf tubes
Load sample(s) into a plate
Degas samples using chamber at the Chemistry Laboratory
Centrifuge plate (Bubbles tend to stick to the plate plastic when out-gassed)
Add silicon lid for Batch setup or Kapton tape for SEC-coflow setup.
Load sample(s) straight on the SAXS beamline for analysis.