Interactions Facility

Capability C of the La Trobe Comprehensive Proteomics Platform (LTU-CPP)

Capabilities

The LIMS Interactions Facility (LIMSIF) provides researchers with a comprehensive range of contemporary platform technologies for the measurement and quantitation of biomolecular interactions in vitro, including protein-protein, protein-nucleic acid, protein-lipid and protein-drug interactions. Key capabilities of the facility equipment include:

  • Determination of binding affinities (KD) and binding kinetics (kon and koff)
  • Measurement of thermodynamic parameters (i.e. stoichiometry, ΔG, ΔH and ΔS)
  • Determination of native hydrodynamic properties (i.e. sedimentation coefficient, molecular weight, diffusion coefficient, and frictional ratio) of proteins and macromolecular complexes in aqueous solution
  • Characterisation of sample homogeneity/heterogeneity, including detection of aggregates
  • Determination of protein secondary structure (i.e. proportion of ɑ-helix, β-strand, β-turn, and random coil)
  • Estimation of thermostability (i.e. Tm and ΔGunfolding)

Equipment

Analytical Ultracentrifugation - Beckman Coulter ProteomeLab XL-A and Aviv Fluorescence Detection System (AU-FDS)

Applications:

  • Measurement of sedimentation coefficient, native molecular weight, frictional ratio (i.e. shape), Stokes radius (Rs) and diffusion coefficient of biomolecules (i.e. proteins, peptides, DNA, RNA and lipids), colloids and nanoparticles
  • Determination of binding affinity (KD) and stoichiometry of homo- and hetero-biomolecular complexes (e.g. dimerisation, trimerisation, tetramerization, etc.)
  • Detection and quantification of aggregation, including higher order assembly (e.g. amyloid fibril formation)
  • AU-FDS optical system enables detection and characterisation of Alexa Fluor 488, FITC or GFP-labelled proteins in the low abundance (i.e. sub-nanomolar) concentration range.

Isothermal titration microcalorimetry - GE MicroCal iTC200

Applications:

  • Determination of binding affinity (KD), stoichiometry (N) and thermodynamic parameters (ΔG, ΔH, ΔS) of protein-protein, protein-lipid, protein-DNA, protein-RNA and protein-small molecule interactions
  • Particularly suited to evaluating protein-drug interactions

Surface Plasmon Resonance - Bio-Rad ProteOn XPR36

Applications:

  • Real-time label free detection of protein-protein interactions
  • Measurement of binding affinities (KD) and binding kinetics (kon/koff)

Microscale thermophoresis

  • Determination of binding affinity (KD) and stoichiometry (N) of protein-protein, protein-lipid, protein-DNA, protein-RNA and protein-small molecule interactions
  • Use of intrinsic tryptophan fluorescence detection for protein-ligand interactions (for ligands that are absent of aromatic fluorophores) using the LabelFree instrument
  • Can employ fluorecently-labelled proteins for determination of protein-protein interaction thermodynamics using the NT.115 RG instrument

Circular Dichroism Spectroscopy - AVIV Model 420 CD spectrometer

Applications:

  • Determination of protein secondary structure
  • Measurement of thermostability

Other major equipment

  • Cary 5 Fluorescence Spectrophotometer (for steady-state fluorescence measurements)
  • Advanced Temperature-Controlled UV/Vis Cary 4000 Spectrophotometer (for protein/DNA/RNA quantitation, enzyme kinetics, and measurement of aggregation via light scattering)
  • Perkin-Elmer Absorbance/Fluorescence ENSPIRE Plate Reader
  • Anton Paar DMA4000 Densitometer and AMVn Viscometer

Contact

For more information about accessing the LIMS Interactions Facility, please contact:
Dr Matthew A. Perugini
Manager of the LIMS Interactions Facility & Director of the LTU-CPP
T: +61 3 9479 6570
E: M.Perugini@latrobe.edu.au