Before Experiment
Before the user group starts an experiment, each member of the group has to complete the beamline user form on the EMBL Hamburg webpage. This form can only be completed ON SITE. It must be printed out, signed and handed in to the administration team.
Safety Training
HASYLAB safety regulations apply to all EMBL beamlines. The safety training can be done in the User Office on the 1st floor. If you need advice, please contact the administration team or your supervising scientist.
Access to EMBL network
In order to connect a laptop to the EMBL network the machine's MAC address must be registered. To do so please register your computer in the EMBL network. You can also use our Computational Facilities and Software.
Sample requirements
The sample concentrations must be determined as precise as possible (accuracy better than 10% is required). The concentrations are necessary to normalize the scattering data. Bradford assays are usually not sufficiently precise, OD measurements @280 nm are better. At the beamline, a Nanodrop spectrophotometer is available. As of November'2008, a refractometer is also installed, which allows one to measure the concentration for proteins lacking aromatic residues.
A single SAXS measurement on X33 requires about 50-60 μl of sample (in the manual filling mode), and the exposure is about 2 minutes. The samples can be damaged by synchrotron radiation during the collection time. A reducing agent (e.g. 2mM DTT) is typically added to the sample before the experiment to diminish the damage/aggregation during the data collection.
An automated sample changer was installed at the beamline in September 2007, providing a more convenient means of operation. To ensure a bubble-free filling of the cell 70-90 μl sample volume is required. It is advised that users utilize the automatic sample changer unless you are severely limited by sample quantity and wish to use the manual filling. Please let us know this in advance, i.e. before going for the experiments.
Each measurement of the macromolecular solution requires two measurements of the corresponding buffer (before and after the sample). The buffers should not contain excessive amounts of additives (e.g. not more than 0.5M NaCl, not more than 5% glycerol, not more than 5 mM ATP etc) unless this is unavoidable. If you need to work with the buffers containing excessive amounts of additives, please consult us in advance.
The users must verify the sample monodispersity (desirably > 90%) prior to coming for the synchrotron radiation measurements e.g. by native gel filtration, ultracentrifugation, DLS etc. If the sample is aggregated, the scattering data will be difficult or even impossible to interpret. Please note that a single band on the SDS gel goes not indicate monodisperse solutions – a single band on the native gel is required!
Typically, for each sample a concentration series (e.g. 1, 2, 5, 10 mg/ml) has to be measured. If your proteins tend to (or may) aggregate at higher concentrations, it is highly recommended to bring the low concentration stocks and concentrate the samples prior to the synchrotron radiation measurements. This can be done using the EMBL wetlab facilities (2nd floor). If the sample is well behaved, one may bring high concentration stocks to dilute them before the measurements.
The cell is cleaned and refilled automatically/manually after each measurement. You should bring sufficient amount (> 10 ml) of matching buffer(s) to dilute the samples in necessary and also to clean the cell.