Protein Interaction
Protein-protein interactions platform offers a large panel of state-of-the-art equipment and expertise in biochemistry and biophysics to perform quality control and functional analyses on protein samples: Analytical Ultra Centrifugation, Microcalorimetry, Microscale Thermophoresis, SEC-MALS and SPR.
Presentation
Microcalorimetry
Microcalorimetry allows the direct, thorough and accurate thermodynamic characterisation of interactions between molecules in solution and of biochemical reactions in general. Two types of calorimeters are available depending on the focus of the study that one wishes to perform:
- Isothermal titration calorimetry (VP-ITC , ITC200 and PEAQ-ITC): principally dedicated to the study of molecular interactions. ITC the only direct method to quantify Kd values in solution without the use any label. ITC is relatively artefacts-free as it is not affected by the optical properties of the samples and it is not limited by the ligand or protein sizes. A direct experiment in the ITC200 instrument detects affinities ranging from nanomolar to low millimolar, although an indirect experiment (competition with a ligand of known affinity) can be used in to extend from pico- to sub-millimolar.
- Differential scanning calorimetry (VP-DSC and auto PEAQ-DSC): principally dedicated to the study of the thermal stability of macromolecules and their complexes. By measuring the variation of heat capacity (ΔCp) as a function of temperature, DSC allows to determine directly the variations of enthalpy (ΔH) and entropy (ΔS) and the melting temperature (Tm) related to each structural transition. DSC systems are powerful tools for characterizing the stability of proteins and other biomolecules that require no assay development, labeling or immobilization. The automated PEAQ-DSC system, available on the platform, offers a high throughput and sensitive analysis of protein stability with low sample consumption.
BIOMOLECULAR INTERACTION
Interactions Analysis with BLI
Based on Bio-Layer Interferometry (BLI), the fluidics-free ForteBio’s Octet® RED96 system is a multi-functional, label-free, real-time analysis instrument. It is ideal for rapidly screening protein-protein, protein-nucleic acids and protein-small molecule interactions. The Octet RED96 system can be used for a wide range of analyses.
System provides up to 8-channel quantitation and kinetic measurements of molecules greater than 150 Da, compatibility with 96-well plates and cooling for temperature control down to 15°C.
Interaction analysis with SwitchSense
SwitchSense is a methodology recently proposed by Dynamic Biosensor. It is based on short DNA nanolevers (48bp or 96bp) that can switch on 24 gold surface spots on a microfluidic chip. The switch of the DNA is mediated by alternating the voltage across the surface (Figure 1, 2). The motion of the levers is tracked in real time (µs sclae) through time-resolved single photon counting using a fluorescence probe signal present on one DNA stand. The complementary DNA strand can be cross-linked to a ligand through amine or thiol coupling or click-chemistry. Upon binding of an analyte, the hydrodynamic friction of the levers is affected and subsequently the movement of this levers. This change is used by the system’s software to describe absolute size/conformation of ligand and complexes and to determine kinetics of the interaction (kon, koff, Kd).
RAPID SAMPLE QUALITY ANALYSIS
Thermal Shift Assay (TSA) or Differential Scanning Fluorimetry (DSF)
This technology measures the thermal stability of proteins by following the fluorescence of a fluorescent dye, the orange SYPRO. A thermal ramp (20 to 95°C) is applied and during protein denaturation hydrophobic areas will appear, the orange SYPRO will attach itself to it and its fluorescence will increase.
To perform these measurements we use the StepOne plus (Applied Biosystem) device.
These rapid measurements (about 1h), carried out in a plate of 96 wells, make it possible to carry out a stability screening of the protein in different buffers or in the presence of different ligands. This technique uses very little material: about 20 µl/ protein well at 0.1 -0.5mg/ml.
Tycho (Nanotemper)
Tycho is used to control the quality of proteins.
A thermal ramp (35 to 95°C) is applied and changes in intrinsic fluorescence detected at 350nm and 330nm (tryptophan and tyrosine) in the protein are measured.
These very fast measurements (about 10 min) are performed in capillaries (6 per run) and require very little sample: 10 µl/capillary of protein at about 0.1 – 0.5mg/ml.
This technology is used to follow protein quality after purification but also to determine the buffer in which the protein is most stable. It is also possible to determine whether ligands (ions, peptides…) have a stabilizing effect on the protein.
SELECTION OF ARTIFICIAL BINDERS (ALPHAREP)
The platform offers generation and production of artificial proteins (alphaRep) that fix with high affinity and specificity any selected target protein. These artificial proteins are also very stable, and easily modifiable (fusion, dimerisation, incorporation of fluorophores, tracers, etc.). The conditions of the delivery depend on the target protein (provided or not) and must make it possible to finance the staff works and reagents/materials used for these projects.
For more information about these services
Contact :
team
Stéphanie MARSIN
Head of Facility
Researcher Engineer
Magali NICAISE-AUMONT
Engineer
(Microcalorimetry
BLI/Tycho/ TSA)
Magali NOIRAY
Engineer
(Microcalorimetry
BLI/Tycho/ TSA)
Michel DESMADRIL
Emeritus Researcher
Agathe URVOAS-CISSE
Researcher
(Binders )
Marie-Béatrice VALERIO-LEPINIEC
Researcher
(Binders )
Philippe MINARD
Researcher
(Binders )
External funding
IBISA
FranceBioImaging
LabexSPS
Morphoscope
Networks
GDR Imabio
RTMFM
SBCF
ISO 9001-NFX50 900 (Qualité)