The scattering intensity of a studied system is given by:
|I(s) ||Scattering intensity of the sample |
|Is(s) ||Scattering intensity of the solution |
|Ib(s) ||Scattering intensity of the background |
|Is,0 ||Intensity of direct beam through the sample |
|Ib,0 ||Intensity of direct beam through the blank sample (e.g. buffer only) |
|D(s) ||Detector response |
|c ||Concentration of the sample |
The resolution of SAXS is about 15 Å.
Four requirements must be fulfilled before performing a SAXS experiment:
- Monodisperse sample (better than 90% needed but better than 95% recommended). Monodisperse samples are essential for studying protein structure via SAXS (low resolution envelope, rigid body analysis, etc.). Polydisperse samples will make the analysis of the scattering data difficult or impossible. The monodispersity of the solution should be checked before you come for your SAXS experiment by dynamic light scattering, native PAGE, ultracentrifugation, etc. Please note that SDS-PAGE results do not indicate monodisperse solutions. It is possible to carry out electrophoresis tests at DLS by prior agreement. Please discuss needs with beamline staff at least 2 weeks in advance.
- A known concentration (1mg to 10mg/ml or as high as the solubility of the protein permits if 10mg/ml is too high). The sample concentration is part of the data reduction and analysis process, so it must be measured very accurately (within 1% accuracy). We would discourage the use of Bradford assays as they are not accurate enough. Optical density measurements at 280nm have been shown to give better results. The method we use utilizes a Nanodrop spectrophotometer, which is available in the B21 Support Lab (Lab 12) and only requires 2ul for each measurement.
- The sample volume per measurement is currently 20µl.
- The buffers should contain less than 0.5M salts. As synchrotron radiation can damage samples, we strongly recommend the addition of a reducing agent (e.g. 2mM DTT) to each sample before the experiment. This will decrease the radiation damage during data acquisition. Reducing agents are not advised for proteins with disulphide bonds. Additional buffer solution is required to flush the cell after each measurement. The buffer for measurement should be the exact buffer that the protein is suspended in. It should also be the same buffer used for dialysis or gel filtration. You may be required to bring your own buffer solution as B21 does not carry all types in Lab 12. Please discuss with the B21 beamline staff prior to arrival.
The scattering intensity of the studied system is obtained by subtracting the background due to parasitic scattering from the slits and/or the windows of the sample holder. In case of molecules in a buffer, the buffer must also be subtracted. This is done by measuring a blank sample, e.g. a sample containing all the components except the molecule of interest.
The intensity of the direct beam through the sample is measured by a photodiode in the beamstop. This is collected automatically, and used to normalise the scattering data. The intensity of the incoming beam is monitored by a beam position monitor just upstream of the sample position.