Equipment available at the beamline X33

• Thermo Scientific NanoDrop ND-1000
• Rudolph Research Analytical J357 refractometer
• Kern ALS 120-4 electronic balance
• Eppendorf Mini-spin microcentrifuge
• Beckman-Coulter Allegra X-22R self-cooling high-speed centrifuge (pcr, 1.5 ml,15 ml,50 ml tubes)
• Gilson auto-pipettes P10, P20, P200, P1000

Major biochemical and biophysical equipment (centrifuge, balance, syringes, pipettes, Nanodrop spectrophotometer, fridge, eppendorf tubes etc) is available at the X33 beamline. A refractometer is also installed, providing a method to measure the concentration of proteins lacking aromatic residues and also for protein complexes. Filtration based concentration devices (e.g. VivaSpin from Sartorius) and dialysis bags are no longer supplied! If samples are to be concentrated on site please bring your own devices.

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.

Superose-6 10/300 and Sephadex-S200 columns are available; superloops are not provided. For the Superose-6 column, the recommended loading concentration is ~10 mg/ml and a loading volume of 200-500 μl. One should then obtain fractions in the 1 mg/ml concentration range and a sample volume of 50-100 μl used for SAXS. 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.

If this service is required users must request beam-time during the week. FPLC is not available on weekends.

Manuals

• NanoDrop manual
• DLS manual

Preparation of BSA for X-ray scattering measurements

Bovine serum albumin (BSA) is a standard that is measured at least once before measuring your samples. The buffer for the BSA (50mM HEPES pH 7.5) and the BSA powder are in the fridge at the beamline.

• Weigh 5 mg of BSA in an eppendorf tube and add 1 ml of buffer.
• Mix by pipetting (try to do it calmly to minimize foam formation).
• Put the supernatant in a fresh tube (it is quite possible that no pellet can be seen, but, to be safe, avoid touching with the tip the bottom of the tube while transferring to the new eppendorf).
• Measure the concentration of the solution at the Nanodrop spectrophotometer at the beamline. You can select BSA in the Nanondrop software to automatically calculate the concentration or in case you want to do it manually the extinction coefficient of BSA is 0.614 (Abs of 1 mg/ml). Usually, you should expect concentrations between 4 and 5 mg/ml.
• Before the measurement, add DTT to a final concentration of 2mM (1M stock aliquots can be found in the -20°C drawer — you can also use them for your samples).