Biophysical Services

Analytical ultracentrifugation (AUC)

AUC is a fundamental tool for studying the size, shape and interactions of molecules and macromolecules in solution.
This tool can be used to perform two types of experiments, referred to as sedimentation velocity (SV) and sedimentation equilibrium (SE).
SV is a hydrodynamic technique and is sensitive to the mass and shape of the macromolecular species. In contrast, SE is a thermodynamic technique that is sensitive to the mass, but not the shape of the macromolecular species. These two measurements provide complementary information, and it is often useful to apply both techniques to a given problem.

 
Beckman-Coulter ProteomeLab™ XL-I  is an ultracentrifuge combined with an optical detection system capable of directly measuring the sample concentration inside the centrifuge cell during sedimentation. This instrument is available in the ZABAM
 

Informational links:
 
Cole et al., 2008
 

 

For experimental design, please contact:
 

Prof. Azem (tel. 03-6409007). azemA@tauex.tau.ac.il 

For operation of the instrument, please contact:
 
Iris bendror (tel. 03-6405100). irisbend@post.tau.ac.il
 

 

Isothermal Titration Calorimetry (ITC)


ITC is a method for determining the affinity of biomolecular interactions. An ITC experiment determines all binding and thermodynamic parameters e.g. KD, stoichiometry, ΔG, ΔH, and ΔS. In every binding event, heat is released or absorbed, this heat is measured by a highly sensitive experimental cell and translated into thermodynamic parameters that includes the binding affinity.
An ITC experiment does not require any labeling of the components and allows direct measurement of sub-millimolar to nanomolar binding constants. Furthermore, no immobilization is necessary and there is a free choice of solvent.
 

Major applications:

 
Characterization of biomolecular interactions such as:

  • Protein – Protein
  • Protein – Peptide
  • Protein – Small Molecules (Drugs)
  • Protein – Nucleic Acids
  • Metal co-Factor – Protein

 
Kinetics & thermodynamics of enzyme catalysis

 

Informational links:

Experimental Summary

Academic Review Paper

 
For experiment design and operation of the system, please contact:
 

Dr. Alex Barbul (tel. 03-6406006). abarbul@post.tau.ac.il

 

 


Light Scattering
 


In a controlled experiment, the scattering of light by molecules is influenced by three variables, molecular weight (MW), concentration and dn/dc, a parameter related to the index of refraction. In many circumstances, dn/dc is constant in aqueous solutions for known concentration and dn/dc, hence one can determine the sample's absolute MW from the scattering of light.

SEC-MALS (Size Exclusion Chromatography – Multi Angle Light Scattering) is a technique that uses HPLC with a size-exclusion column (separation between 10 – 150 KDa or between 10 – 1000 KDa) to separate different sizes molecules together with MALS system to measure the molecular weight of the separated molecules and a refractive index detector to measure the concentration at each measuring point. For large molecules (> 10 nm)  one can also measure the radius of gyration (Rg). 

There is also possibility to work in a batch mode (without the SEC) to measure the average molecular weight of all the solutes in a solution. In this mode one can also measure the second virial coefficient, A2, or the dn/dc of molecules in solution. A2 is a measure of the intermolecular interactions (non-ideality).

CG-MALS (Concentration Gradient – MALS) is a technique which can be used to measure self and hetero association of molecules in solution. It can determine parameters such as KD, A2 and for slow kinetics also kon and koff.it provides also the true stochiometry of the interacting molecules. In this technique, one measures the light scattering of a molecule in different concentrations. The system is fully automated with syringe-mixer.
 

For experiment design and operation of the system, please contact:
 

Hezi Sasson (tel. 03-6406211). sassonye@post.tau.ac.il

 

 

 

 

 

Tel Aviv University, P.O. Box 39040, Tel Aviv 6997801, Israel
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