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Protein Expression:

- Platforms

- Cloning

- Screening

Protein:

- Purification

- Characterisation

- Crystallisation

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Protein Facility Support and Services

The NKI Protein Facility is open available for Academic scientists. Both NKI- as well as external scientists can apply for projects.

A list of support and services is given below. For more details please check the individual pages. If your request is not available from the list, please contact us to discuss the possibilities.

Initial experiments can be performed by the Facility members, however the aim of the facility is to assist researchers with performing their own experiments. For each individual project it will be discussed up to what level the Facility will execute the experiments and when the applicant has to provide hands-on support. A number of experiments will be provided by the Facility as a service, e.g macromolecular crystallisation.

Construct Design

  • Construct Design using CCD (Crystal Construct Designer) web application for selecting optimal constructs.

  • Recommendation for cloning, including assistance with LIC (Ligation Independent Cloning).

  • A series of LIC vectors is available from the Facility

Protein Expression

  • Protein expression tests in E. coli T1 Phage-resistant cells at small scale.

  • Large scale protein expression in E. coli T1 Phage-resistant cells, up to 6 liters per batch.

  • Production of Baculovirus viral stock solutions.

  • Expression of proteins from baculovirus in insect cells, up to 4 liters per batch.

  • Mammalian expression system, using HEK293 and derivatives like Glycosylation deficient HEK293 strain (GnT-) and HEK293 Flp-In cells for creating stable cell lines.

Protein Purification

  • Small scale purifications with affinity resin (Ni-NTA-, Glutathion beads etc.).

  • Äkta FPLC systems are used for protein purification by automated column chromatograpgy.

  • A variety of columns are available, including Anion-, Cation-, Size Exclusion (Gelfiltation)- and Desalting columns.

Biophysical Characterisation

  • Isothermal titration calorimetry (ITC) for studying macromolecular interactions, including protein-protein, protein-small molecule, protein-DNA and protein-peptide interactions.

  • Surface plasmon resonance (SPR, BIACORE) for analysis of interaction between protein-protein, protein-DNA and protein-peptide interactions.

  • Fluorescence Polarisation for binding analysis of fluorescent-labeled molecules to proteins

  • Analysis of protein thermal stability using thermofluor assay.

  • Identification of protein multimerisation / complex formation via molecular mass determination using multi angle laser light scattering (MALLS) coupled to size exclusion chromatography

Crystallisation

  • High-throughput screening of crystallization conditions in 96-well format (vapour diffusion) in nanoliter volumes.

  • Screening of crystallization conditions using TOPAZ microfluidics system (96-well format).

  • Optimization of crystallization conditions.

NKI Protein Facility                                                                                                                                                                                                         Last updated: 13 Aril 2012

NKI-AvL

Plesmanlaan 121

1066 CX Amsterdam

The Netherlands