Molecular Biotechnology Core
The Molecular Biotechnology Core offers biomolecular interaction analysis and equilibrium and kinetic measurements using Biacore S200, Isothermal Titration Calorimetry and Microscale Thermophoresis, and CD Spectroscopy.
The Core provides a wide range of high quality services of surface plasmon resonance (SPR) and other biophysical technologies. Biomolecular interactions are quantified using Biacore S200 and NanoTemper (NT, LabelFree and NT.115), while isothermal titration calometer (iTC MicroCal 200) is used for measuring the thermodynamic parameters (kinetics, enthalpy, entropy and number of binding sites) of biomolecular interactions. Jasco 815 CD spectrophotometer is capable for analysis of secondary structure of proteins in solution and protein folding and confirmation changes.
- Surface Plasmon Resonance
- Isothermal Titration Calorimetry
- Circular Dichroism Spectroscopy
- Microscale Thermophoresis
A Circular Dichroisms (CD) Spectropolarimeter (Model J-815 from Jasco) is a type of light absorption spectroscopy that can provide information on the structures of optically active biological macromolecules. CD spectra of proteins between 250 and 185 nm can be analyzed for different secondary structural types such as, alpha helix, parallel and antiparallel beta sheet, turn and other random structures.
The CD spectroscopy is a shared resource and is available to researchers of Cleveland Clinic Foundation and neighboring Institutions. The details about CD spectroscopy are as follows:
- Model J-815 Spectropolarimeter (CD and ABS measurement modes) with computer controlled data acquisition; wavelength range 163-900 nm.
- Single position Peltier system with Fluorescence capability.
- Dual range titration system for automated pH, ligand and denaturant titrations.
- Scanning Emission Monochromator allows spectral EM scans simultaneous with CD measurement (wavelength range 200-750 nm)
- Temperature-controlled circulating water bath (Julabo)
CD Spectropolarimeter can be used for the following applications:
- Secondary structure analysis of proteins in solution
- Protein folding and conformational studies
- DNA/RNA interaction studies
- Temperature controlled kinetic studies
- Thermal denaturation profiles of aqueous proteins
Isothermal Titration Calorimetry
What is ITC?
Isothermal Titration Calorimetry (ITC) is the gold standard for measuring biomolecular interactions. ITC simultaneously determines all binding parameters (n, K, δH and δS) in a single experiment information that cannot be obtained from any other method. When substances bind, heat is either generated or absorbed. ITC is a thermodynamic technique that directly measures the heat released or absorbed during a biomolecular binding event. Measurement of this heat allows accurate determination of binding constants (KB), reaction stoichiometry (n), enthalpy (δH) and entropy (δS), thereby providing a complete thermodynamic profile of the molecular interaction in a single experiment. Because ITC goes beyond binding affinities and can elucidate the mechanism of the molecular interaction, it has become the method of choice for characterizing biomolecular interactions.
- Characterization of molecular interactions of small molecules, proteins, antibodies, nucleic acids, lipids and other biomolecules.
- Lead optimization.
- Enzyme kinetics.
- Assessment of the effect of molecular structure changes on binding mechanisms.
- Assessment of biological activity.
Interactions between any two molecules can be studied with ITC, including:
- Protein-small molecule
- Small molecule-small molecule >
Microscale thermophoresis (MST) has been recently emerged as a powerful technique for quantifying molecular interactions. It is highly sensitive and can quickly yield detectable signals in response to interaction-induced changes of molecular properties, including size, charge, hydration shell and conformation. This technique is based on thermophoresis, which is defined as temperature gradient-induced directed motion of molecules. In a typical MST experiment using the Monolith NT.115, a temperature gradient is induced by an infrared laser and the resulting change in molecular movement is analyzed by fluorescence. Thus, one of the binding-partner must be a biomolecule with covalently attached fluorophore or a fluorescent fusion protein like GFP. However, MST experiments can also be performed in a label-free setting using intrinsic fluorescence of protein in another Monolith model (NT.LabelFree) The interactions can be measured in complex biological fluids like cell lysate and serum without sample purification, which is much needed for other techniques. It also permits studying of the interaction of small molecules and proteins with ease or membrane proteins stabilized in buffers of choice. Thus its high adaptability over other techniques renders it unique and unparalleled.
MST Application Includes:
- Affinities between biomolecules including proteins, DNA, RNA, peptides, and small molecules
- Stoichiometry of an interaction
- Dissociation constants of multi-component reactions
- Competition between substrates and inhibitors to an enzyme
- Interactions of biomolecules with nanoparticles, vesicles and viruses
- Surface modifications of nanoparticles
- Recognize different binding sites on a target molecule of interest
- The stability of biomolecules in blood, blood serum and blood plasma
- Adsorption of small molecules to lipid membranes or plasma proteins
- Protein aggregating compounds/conditions
Surface Plasmon Resonance (SPR)
Surface Plasmon Resonance (SPR) has been used to monitor macromolecular interactions in real time. The Biacore S200 system is an instrument that uses SPR technology for measuring the interactions of macromolecules with each other, and with small ligands. One of the interacting molecules (ligands) is immobilized, for example, on carboxymethylated dextran over a gold surface, while the second partner (analyte) is captured as it flows over the immobilized ligand surface. Most ligands can be directly immobilized onto the surface of the chip via amino groups, carbohydrate moieties, or sulfhydryl groups. Others are immobilized indirectly through the use of biotinylation of the ligand (such as biotinylated peptides or oligonucleotides), or through immobilized monoclonal antibodies (such as anti-GST). Typical amounts of a protein ligand needed for an immobilization reaction is about 1 µg. The immobilized ligands are remarkably resilient and maintain their biological activity.
Biacore can be used for measuring the binding constants of macromolecular interactions (protein-protein, nucleic acids - protein, protein-ligand, etc.) and in many other applications such as, epitope mapping, ranking antibody affinities, concentration measurements, characterization of mutant proteins and Ligand fishing.
The bound analytes can be stripped from the immobilized ligand without affecting its activity to allow many cycles of binding and regeneration on the same immobilized surface. Interaction is detected via SPR, in real time, at high sensitivity, without labeling. The equilibrium rate constant KD (affinity) is measurable in the range of fM to mM. Because the same affinity may reflect different on-rates and off-rates, this instrument excels over most other methods of affinity measurements in that it measures on-rates (ka in the range of 103 to 3 × 109 M-1s-1 for proteins and 103 to 5 x107 M-1s-1 for LMW) and off-rates (kd of 10-5 to 2 s-1).
The instrument and services are available on a fee-for-service basis to Cleveland Clinic researchers as well as investigators from Case Western Reserve University and other area institutions.
The Molecular Biotechnology Core offers consultation and services in the areas of biomolecular interaction analysis and equilibrium and kinetic measurements, and secondary structure determination.
The Core offers training and facilitates biomolecular interactions analysis using the Biacore S200 system, based on the principle of Surface Plasmon Resonance (SPR). The Biacore S200 is highly sensitive and used for measuring the interactions of macromolecules with each other or with small ligands, and for equilibrium and kinetic measurements, competition assays, and epitope mapping. The core provides hands-on training on both Biacore S200 and the S200 Evaluation software to users.
The Core offers training and assists researchers in using Isothermal Titration Calorimetry (ITC Microcal 200). ITC is a thermodynamic technique that measures the heat released or absorbed during a biomolecular binding event. It allows accurate determination of binding constants (KB), reaction stoichiometry (n), enthalpy (δH) and entropy (δS), thus providing a complete thermodynamic profile of the molecular interaction in a single experiment.
The Core provides training and assists researchers in using Microscale Thermophoresis (MST). It is a powerful technique for quantifying molecular interactions. This technique is based on thermophoresis, which is defined as temperature gradient-induced directed motion of molecules. In experiments using the Monolith NT.115, one of the binding-partner must be a biomolecule with covalently attached fluorophore or a fluorescent fusion protein like GFP. MST experiments can also be performed in a label-free setting using intrinsic fluorescence of protein in another Monolith model (NT.LabelFree). It also permits studying of the interaction of small molecules and proteins with ease or membrane proteins stabilized in buffers of choice. Thus its high adaptability over other techniques renders it unique and unparalleled.
The Core maintains and provides training to use CD spectroscopy. CD Spectroscopy is a valuable instrument for rapid analysis of structural and conformational changes in a protein upon perturbation by mutation, temperature, pH and buffers. CD spectra arising due to peptide bond transitions in the "far-uv" spectral region (190-250 nm) provide information on secondary structure of protein, while CD spectra of proteins in the "near-uv" spectral region (250-350 nm) provide information on certain aspects of tertiary structure.
Molecular Biotechnology Core