Planning Your Experiment

If you would like to collect high quality SAXS data to derive accurate and absolute structural parameters of a monodispered particle or for molecular modelling, we recommend that you plan to use size exclusion chromatography coupled SAXS (SEC-SAXS). If it is important that you accurately control the composition of your sample while systematically varying some parameter i.e. a titration series of a ligand, you should plan to load your samples directly onto the beamline via our liquid handling robot. This mode is typically referred to as "static" or "robot" mode. Since it can be difficult to demonstrate that samples collected in static mode are homogenous, it is generally best to interpret the relative structural changes across the series rather than the absolute value of the parameters.

Protein samples are typically 35 μl in volume and are at concentrations between 0.1 and 5 mg/ml. Below are some guidelines for optimising your sample. If these cannot be met then measure your non-ideal sample, you might be lucky and the results will help in optimising a future experiment. Before planning and running a large, complicated experiment such as a multifactorial screen, it is a good idea to make a simple measurement of your sample to check its suitability. Our mail-in service is useful for pre-screening and optimising samples prior to more complex experiments.

A typical robot experiment would be as follows:

1) 35 µl buffer

2) 35 µl sample at 2 mg/ml

3) 35 µl sample at 1 mg/ml

4) 35 µl sample at 0.5 mg/ml

5) 35 µl sample at 0.25 mg/ml

6) 35 µl sample at 0.13 mg/ml

7) 35 µl repeat buffer

Volume

By default the liquid handling robot loads the sample to the beam position and then flows the sample past the beam at 1 µl.s^-1 while collecting data to limit radiation damage. Increasing the volume of the sample will allow for longer collection times improving signal to noise. The minimum recommended sample volume is 20 µl, the maximum sample volume is 100 µl and the default is 35 µl.

Buffer

It is critical that with each robot sample an accurately matched buffer is supplied. If a size exclusion column was used as the last step in purification some of the flow through taken from close to the sample peak is likely to work well. Some of the flow through from a pre-rinsed spin concentrator is likely to work well if you concentrated your sample in this way. Dialysing your sample and taking some of the dialysate as a buffer blank is the method most likely to work.

Buffers will be optimised so that the sample is most stable. Organic buffers such as Tris give better protection from radation damage than phosphate. Adding 1-2% glycerol or sucrose is also useful for preventing radiation damage. Including some salt (i.e. 100 mM) is helpful for avoiding inter-particle correlation effects that can effect data quality. Detergents or lipids are best avoided if at all possible as they form larger structures such as micelles that interact with sample making them extremely difficult/impossible to remove by blanking. Detergents used at low concentrations and in earlier stages of the purification proceedure can co-elute and concentrate along with the sample.

A typical protein SEC-SAXS sample is 50 µl at 5-10 mg/ml. If possible take 250 mls of running buffer, which is often taken in a 50 ml falcon tube as a 10x stock. Plan a minimum of 1 hour to equlibrate a column into a new buffer and 35 minutes for each sample that you want to run.

Buffers

Buffers will be optimised to stabilise the sample. Including 1-2% glycerol or sucrose helps reduce radation damage. Including some salt (i.e. 100-150 mM) will help reduce interation between the particle and the column and inter-particle correlation effects that could reduce data quality. Detergents/lipids can form higher order structures that can interact with the sample and are best avoided.

If the buffer contains reagents that are prohibitively expensive or limited then you could limit the buffer volume to a bare minimum of 28 mls for one run (10 mls buffer to cover the HPLC buffer line filters, 10 mls to purge the HPLC pump, 5 mls for column equilibration and 3 mls per run). Otherwise it is better to bring plenty of buffer i.e. 250 mls.

If you have queries please speak to a member of the beamline team.

Useful phone numbers during planning or during your visit (external dial is 01235 77 XXXX):