The Protein Facility can train users to make use of the equipment. This way the researcher has control over the timing and proceeding of their project, as well as enjoying an educational component. Obviously, the Protein Facility will still be available for advice and tips if necessary. Before starting a project we generally invite the potential user for a take-in meeting in which they can briefly explain the project and we come to a project plan.
The training and guidance will be limited by the project and can include usage of the FPLC machines, any of the biophysical analysis machines or any combination. . We are equipped for the production of most types of target proteins (see Equipment) and can benefit from being embedded in the department of Chemical Immunology to set up your analysis assays.
If the intake meeting shows that there is very little gain (for either side) to pursue the extensive training, the Protein Facility can also offer to produce the required protein. This happens for instance if it is a one-off production of a well-described enzyme or if time constraints come into play.
Contact us for more information.
The Protein Facility is equipped with all requirements for heterologous expression and purification of recombinant proteins. Furthermore, a variety of biophysical techniques is available to determine the protein quality, but also to perform activity or interaction experiments.
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 liters.
- Baculoviral expression. We also have a variety of expression constructs and the means to generate BacMids for protein production using Sf9 insect cells.
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.
- 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.
Contact and Team
Robbert Q. Kim
Tel: +31 (0) 71 526 8701
|Robbert Kim||Angeliki Moutsiopoulou|
Department of Cell and Chemical Biology (CCB)
Leiden University Medical Center (LUMC)