Equipment

Protein expression systems

• Bacterial expression. We routinely test a variety of expression strains with all common expression tags. Our expression setup allows for expressions of up to 80 litres.
• Baculoviral expression. We also have a variety of expression constructs and the means to generate BacMids for protein production using Sf9 insect cells.

Protein purification

We have a range of ‘standard’ affinity beads and columns, compatible with our expression plasmid set. These can use in either a gravity flow set up as well as using our Bio-rad NGC FPLC systems in a continuous flow.

• Affinity purification. His-tag (Ni-NTA or TALON), GST-tag or Strep-tag purification is carried out regularly
• Ion exchange. Including cation (ResourceS, HiTrap S, PorosS) or anion (ResourceQ, HiTrap and PorosQ) exchange or Heparin columns
• Size exclusion. Both small (10/30) and large (16/60, 26/60) preparative scale columns with various bead size (Superdex75, Superdex200/SEC650, Superose6) are available for use on the NGC. Analytical runs: see below.
• Others. Various other, specialized columns for hydrophobic interactions or chitin-binding are available.

Biophysical analysis

• Sample purity. Generally this is tested using SDS-PAGE, and can, in some cases, also be addressed using HRMS at the Ovaa lab.
• TM measurements. Using the Tycho NT 6.1 melting curves for protein quality can be obtained.
• Analytical chromatography. Most purification columns are available on our ÄKTAµ setup, to allow screening using low protein amounts.
• MALS. We have a Wyatt setup for Multi Angle Light Scattering, coupled to size-exclusion to obtain protein size and stoichiometry.
• DLS. We have a Wyatt Nanostar for batch Dynamic Light Scattering measurements.
• ITC. For isothermal titration calorimetry we have a TA Instruments Affinity available with nanolitre cell volume (190 µL)
• Spectrophotometric plate reader. For fluorescence based assays we have the BMG LabTech ClarioStar, capable of tuning to any wavelength (230-800 nm).
• BLI. On our Sartorius Octet96 we are able to perform biolayer interferometry experiments, allowing us to determine on- and off-rates (and thus K­D) for biomolecular interactions.