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Neurotargeting

Methods

To study and characterize proteins it is important to follow it all the way from DNA until modified protein as it exists in the cell. To do so, there is a jungle of methods to use. Here we list some of the methods that we use:

Protein production

On DNA level:

  • ”Seam-less” cloning methods
  • Gateway cloning methods
  • Site directed mutagenesis (PCR-based)
  • In vitro transcription
  • SNP genotyping and sequencing (collaboration with Center of Medical genetics and Molecular biology)

 

On Protein level:

  • Expression and purification of recombinant proteins using E. coli, Pichia pastoris,and Spodoptera frugiperda
  • In vitro translation
  • Denaturing and non-denaturing polyacrylamide gel electrophoresis (SDS- and native-PAGE)
  • In vitro RNA-protein interactions
  • In vitro protein-protein interactions
  • Various pull-down interaction assays
  • Phosphorylation studies (including MS, carried out by PROBE)
  • Immunostaining
  • Functional assays (activity, stability, interactions etc)

Cell culture

  • Expression of recombinant proteins in different cell lines such as HEK293, HELA, SH-SY5Y
  • Stimulation, inhibition and stability assays
  • Confocal and fluorescence microscopy
  • Functional assays (activity and interactions etc)

Biophysical Methods

  • Solution small-angle X-ray scattering and diffraction (SAXS/SAXD)
  • Circular dichroism spectropolarimetry (CD)
  • Synchrotron radiation circular dichroism (SRCD)
  • Oriented circular dichroism (OCD)
  • Surface plasmon resonance (SPR aka Biacore)
  • Isothermal titration calorimetry (ITC)
  • Differential scanning calorimetry (DSC)
  • Atomic force microscopy (AFM)
  • Fluorimetric assays, like intrinsic Trp-fluorescence
  • Turbidimetric assays
  • UV-VIS absorption assays
  • Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS, collaboration in Oulu)
  • Dynamic light scattering (DLS)
  • Thermal shift assays/Differential scanning fluorimetry (aka Thermofluor or DSF)
  • Various neutron methods (diffraction, reflectometry, small-angle scattering, EINS, QENS, etc.)
  • Transmission electron microscopy (negative stained samples, immunostained samples)
  • Cryo electron microscopy (Collaboration)
  • Static light scattering (SLS/SEC-MALS)

Structural Biology

  • Macromolecular X-ray crystallography
  • Solution nuclear magnetic resonance spectroscopy (NMR, collaboration)