Equipment available at the P12 beamline

• Thermo Scientific NanoDrop ND-1000
• Eppendorf Mini-spin microcentrifuge
• Pipettes

Equipment in the User Lab next to the beamline

• Anton Paar Abbemat 550 refractometer
• Wyatt DynaPro NanoStar DLS
• Kern ALS 120-4 electronic balance
• Beckman-Coulter Allegra X-22R self-cooling high-speed centrifuge (PCR, 1.5 ml, 15 ml, 50 ml tubes)
• Eppendorf Centrifuge 5430 (non-cooling)
• Gilson auto-pipettes P10, P20, P200, P1000

The refractometer provides a method to measure the concentration of proteins lacking aromatic residues and protein complexes.

If for some reasons the buffer cannot be prepared in advance, ddH₂O and a vacuum filtering device is found on site (User Lab, SPC kitchen).

Filtration based concentration devices (e.g. VivaSpin from Sartorius) and dialysis bags are not supplied. If samples are to be concentrated on site please bring your own devices.

Online sample purification and characterization

The quality of SAXS data can be increased significantly by an online purification system allowing immediate data acquisition after the individual components elute from a size exclusion column (SEC). Moreover, the analysis of the SAXS data is greatly facilitated if the sample is characterized biophysically by complementary techniques. We, therefore, offer the possibility to use a multi-detector SEC system which allows the determination of the concentration as well as the molecular weight of the eluting fractions. It is, therefore, possible to use this system to characterize the oligomeric states of individual proteins and analyse the formation/dissociation of complexes.

Please inform your local contact prior to your visit if you plan to use this set-up.

Equipment available at the SPC facility

Additional wetlab based needs can be met at the integrated sample preparation and characterization (SPC) facility (building 48e, ground floor) which provides chemicals, DLS spectrometer, FPLC system. If access to the SPC facility is required, an email to spc@embl-hamburg.de with a copy to the local assigned contact must be sent at least 14 days in advance.

FPLC

Prior to measurement users have the possibility to use the GE ÄKTA purifier FPLC system which is located at the SPC facility. A gel-filtration run is advised for difficult samples to separate aggregates and higher oligomers from the protein to be studied. It is also possible to use this system to analyse fractions from gel-filtration and characterise oligomeric states of individual proteins or the formation/dissociation of complexes.

Columns and superloops are not provided. A typical run is approximately 2 hours (including column equilibration). Users are encouraged to bring 1 litre of buffer for column equilibration and the subsequent run, however, when this is not possible buffers can be prepared on site.

Sample storage

• Refrigerator in the P12 control hutch (4°C with -20°C shelf, to use during the day of the experiment)
• Ice machine is in the SPC kitchen
• 2 shelves are available for SAXS samples in the cold room (4°C) in the SPC area
• Storage in the -80°C cupboard in the SPC lab
• Cooling cabinet (approx. -50°C) near the P12 control hutch

Access to the cold room and -80°C storage upon arrival

From the EMBL main building (25a), you need to cross the street and walk in the direction of 48e. Use the staircase on the right side of the building to reach the 1st floor, you will find an entrance there (which in contrast to the main entrance is open on weekends and after working hours). The facility is on the ground floor.

The SPC kitchen room (L003) which contains an ice machine and the cold room (L008a) next to the SPC always remain unlocked.

Manuals

• Preparation of BSA
• NanoDrop manual
• Refractometer manual
• DLS manual
• Wyatt-MALLS-DLS