- Protein facility - NKI


The NKI Protein facility is a protein production and characterisation facility at the Division of Biochemistry, The Netherlands Cancer Institute (NKI).


The NKI Protein Facility is part of the Instruct-NL National Instruct Centre in the Netherlands and provides support for the production, purification, biophysical characterisation and crystallisation of proteins. It is accessible to both internal (NKI) and external research groups. In particular researchers who don't have the experience or the facilities to produce and analyse proteins can request for assistance with the design and performance of the experiments. Some experiments (e.g. high throughput macromolecular crystallisation) will be performed by the facility members and are offered as a service. A particular strength of the facility is in the characterisation of macromolecular interaction with a multitude of biophysical methods. Importantly, to ensure a smooth application process, we strongly recommend any users to contact us before submitting an application.

 

Construct design and cloning

Expression of soluble,functional proteins is dependent on many different factors. The facility can assist in the design of selective protein fragments using structural- and bio-informatics tools to increase the chance for success.

 

Protein production and purification

The facility has platforms for protein expression in E. coli (large-scale incubators at different temperatures), in insect cells using the baculovirus system, and for protein expression in mammalian cells (large roller-bottle incubator with a capacity for 90 bottles).

A variety of chromatography steps - including affinity, ion exchange and size exclusion chromatography - can be performed on different Akta and BioRad systems.

 

Biophysical characterisation

The facility offers a variety of biophysical techniques to characterise macromolecular properties and interactions. These include:

 

  • The OPTIM1000 system for label-free measurement of thermal stability and aggregation
  • A Q-PCR device for measuring protein thermal stability by Thermal Shift Assay (Thermofluor)
  • Multi-angle laser light system (MALLS) coupled to size exclusion chromatography for molecular weight determination in solution
  • A Biacore T200 instrument for measuring interactions by surface plasmon resonance, using an immobilised component
  • A VP-ITC to measure interaction between unlabelled macromolecules and/or ligands using Isothermal Titration Calorimetry
  • A multifunctional fluorescence plate reader (e.g. for fluorescence intensity or polarisation assays)
  • A microscale thermophoresis device for analysing interactions by heat induced motility of labeled macromolecules
  • A stopped-flow system with multiple fluorescence detectors for measuring fast kinetics (msec scale)

Crystallisation screening

The facility has access to an automated high-throughput crystallisation screening platform. Crystallisation conditions can be screened for in 96-well format in 100-200 nanoliter droplets. Plates are prepared by (nanoliter) liquid handling robots and crystallisation trials are visualized over time by automated imaging systems at 4 °C and 20 °C; results are accessible via web-servers.

NKI Protein Facility

Plesmanlaan 121

1066CX

Amsterdam

Netherlands

Protein facility - NKI website

Visitor information

NKI is currently available for remote access only due to COVID-19 restrictions. For information on the availability of specific technologies, view the Technology Availability List or contact support@instruct-eric.eu. Please check here regularly for updates.

 

How to find us

The Netherlands Cancer Institute/Antoni van Leeuwenhoek hospital (NKI/AvL) is conveniently situated at the south-west side of Amsterdam. From Schiphol airport, it is about 15 minutes drive to the institute (bus or car) or 7 minutes by train (to Amsterdam Lelylaan station, followed by a 15 minute walk). From the institute it is easy to connect to Amsterdam city centre via tram, bus or train. More details can be found at the NKI website.

Flagship Service/Technology at Protein facility - NKI:

A biophysical characterisation pipeline for judging the suitability of macromolecular samples for structural analysis.

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Other Services/Technologies at Protein facility - NKI:

The Protein Facility of the Netherlands Cancer Institute (NKI) in Amsterdam has a strong record in structural biology using macromolecular X-ray crystallography, Electron Microscopy and sample preparation, and is also extremely well-equipped for studying molecular biophysics.

We are very experienced in macromolecular crystallization and offer to our users:

- Robotics for preparation of nanoliter crystallization drops in 96-well format.

- Storage of crystallization plates at 4°C or 20°C.

- Screening and ranking of crystallization conditions in 96-well format (TOPAZ, AutoInspex station).

Solving crystal structures is beyond the scope of the facility!

 



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The Protein Facility of the Netherlands Cancer Institute (NKI) in Amsterdam has a strong record in structural biology using macromolecular X-ray crystallography, Electron Microscopy and sample preparation, and is also extremely well-equipped for studying molecular biophysics.

With respect to Molecular Biophysics we offer to our users fluorescence-based fast kinetics and high-throughput experiments, ITC, MST, MALS, SPR and Thermal Shift Assays.

 



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The Protein Facility of the Netherlands Cancer Institute (NKI) in Amsterdam has a strong record in structural biology using macromolecular X-ray crystallography, Electron Microscopy and sample preparation, and is also extremely well-equipped for studying molecular biophysics.

We are very experienced in protein production using insect and mammalian cells, and assist users with the design of their expression construct in terms of solubility, dopmain boundaries, etc. We offer a variety of exprssion vectors, including a set of LIC vectors (Ligand Independent Cloning) for protein expression in insect cells and mammalian cells. For expression in insect cells we have SF9 and SF21 systems for expression of intracellular and extracellular proteins.Our mammalian expression system has been optimized for HEK-293T cells, and we also have the glycosylation free, gnT1-deficient HEK293S variety.

Protein expression in bacteria (E. coli) is beyond the scope of the facility!

 



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Scientific Contacts
Anastassis Perrakis
Anastassis Perrakis

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Patrick Celie
Patrick Celie

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Hans Wienk
Hans Wienk

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Admin Contacts
Patrick Celie
Patrick Celie

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Hans Wienk
Hans Wienk

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Technical Contacts
Patrick Celie
Patrick Celie

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Alexander Fish
Alexander Fish

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Hans Wienk
Hans Wienk

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