The facility provides support for efficient design and cloning of genes into expression vectors.
- E. coli: Multiple Multritron (Infors-HT) shakers with a capacity of 8 liter bacteria culture per incubator are available for temperature-controlled bacterial growth and expression of proteins. Bacterial cells can be pelleted in a ThermoFisher Scientific Sorvall large capacity centrifuge (12 liter culture).
- Sf9 Insect cells: Proteins can be expressed in sf9 insect cells via the Baculovirus system. The facility has access to three temperature-controlled shakers for sf9 suspension cultures and a CASY cell-counter for monitoring cell viability, growth and viral infection. Conditioned medium and/or cells can be harvested by centrifugation (see E. coli)
- Mammalian cells: HEK293 Cells can be grown (adherent) in plates, T-flasks and roller bottles. Two temperature- and CO2- controlled incubators are available for growing cell cultures in plates. A large scale Roller bottle incubator (Incudrive) is available for culturing cells in roller bottles.
Three cell-disrupting systems are available at the facility to prepare cell lysates:
- A QSonica Sonicator is used to break cells by pulses of ultrasound.
- A Emulsiflex (Avestin) high-pressure homogenizer to disrupt cells.
- A Retsch Mixer Mill to physically grind cryogenic-cooled cells by vibration of the sample with metal balls.
When proteins need to be purified from conditioned medium (antibodies or secreted proteins), medium is first concentrated from several liters to 100 – 300 ml in a Fresenius F40S hollow fiber dialyser to facilitate follow-up purification procedures.
The facility has access to various chromatography systems for protein purification, including:
- BioRad NGC system for large scale protein purification
- Äkta Micro and NGC-micro systems for small scale (analytical) purifications
- Systems can be equipped with a large variety of columns, including affinity-, ion-exchange- , Size Exclusion- and hydrophobic- chromatography columns.
A selection of methods/instruments is available for protein quality control and analysis, including:
- SDS-PAGE analysis
- NanoDrop protein quantification
- SEC-MALS (Akta FPLC-Wyatt): Static light scattering coupled to size exclusion chromatography for measuring molecular weight of protein species separated by size (stoichiometry/aggregation analysis)
- NanoTemper Prometheus NT. 48 nano-DSFT (nano-Differential Scanning Fluorimetry): Measuring protein thermal stability using intrinsic tryptophan fluorescence and light scattering as read out for protein unfolding and aggregation, respectively
The facility houses a selection of instruments for exploring molecular interactions of proteins, including:
- Biacore T100 instrument for chip-based Surface Plasmon Resonance experiments for examination of binding kinetics and molecular affinity.
- MicroCal VP-ITC (Isothermal Titration Calorimetry) instrument to assess thermodynamics of molecular interactions in solution via heat exchange during binding of molecules in a titration series.
- Nanotemper Monolith system to record Micro-Scale Thermophoresis (MST), a phenomenon based on change in mobility of protein (-complexes) upon short laser-induced heat induction.
- BMG Labtech Plate readers (FLUOstar and CLARIOstar) for flurescence- and fluorescence polarization (FP)-based assays.
- HI-TECH Stopped-flow system for measuring fast (millisecond)kinetics using fluorescence (polarisation).
The facility host a high throughput crystallisation platform for screening and optimisation of protein crystallisation conditions by vapour diffusion:
- Cybio-Selma semi-automatic pipetting robot (96-tip head) for transfer of crystallisation reagents into 96-well crystallization plates.
- SPT Labtech Mosquito for preparation of crystallisation droplets in nanoliter scale in 96-well plates.
- Two Rockimager (Formulatrix) systems for storage and automated imaging of crystallisation plates at 4 °C and 20 °C, respectively.
- Formulator (Formulatrix) for preparation of customized crystallisation solutions for optimisation of crystal growth conditions in 96-well format.