Instruct-NL - Protein facility - NKI


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

The NKI Protein Facility (external website) 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 analyze 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. Important: 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 characterization

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 polarization 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).

Crystallization screening

The facility has access to an automated high-throughput crystallization screening platform. Crystallization conditions can be screened for in 96-well format in 100-200 nanoliter droplets. Plates are prepared by (nanoliter) liquid handling robots and crystallization 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

http://www.proteinfacility.nki.nl

Visitor information

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 min. 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:

http://www.nki.nl/topmenu/contact/

Scientific Contacts
Anastassis Perrakis
Anastassis Perrakis

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Patrick Celie
Patrick Celie

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Admin Contacts
Patrick Celie
Patrick Celie

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Magda Stadnik
Magda Stadnik

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Technical Contacts
Patrick Celie
Patrick Celie

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Magda Stadnik
Magda Stadnik

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Alexander Fish
Alexander Fish

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