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Laboratory of Biochemistry and Functional Proteomics

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Instrumentation  

 

Nuclear Magnetic Resonance Fast-Field-Cycling Relaxometer

Fast Field Cycling Relaxometry (FFCR) is a NMR technique used to determine the longitudinal relaxation time (T1) over a range of magnetic fields spanning about 6 decades, from about 10-6 up to ~1 Tesla. Since the very beginning of Nuclear Magnetic Resonance there had been great interest in relaxation phenomena, including their field dependence. Starting in the fifties, methods for acquiring T1 dispersion profiles (plots of longitudinal relaxation time as a function of field intensity at a constant temperature) had been used.

The FFC magnet

 

This approach, known as Fast Field Cycling, has been tried with success in several laboratories (S. H. Koenig and R. D. Brown at IBM, F. Noack and at the University of Stuttgart). The few pioneering groups who had access to this type of instrumentation exploited the tight link between NMR relaxation phenomena and molecular dynamics to explore at least three possible application fields: the hydration of paramagnetic metal ions and organometallic complexes, the dynamics of liquid crystals, and the dynamics of proteins. The wealth of information obtained from these studies, together with the awareness that many more application fields remain totally unexplored, led to the recent dramatic increase of interest in FFC NMR relaxometry. These early studies have confirmed the theoretical work of many distinguished physicists, linking NMR relaxation phenomena to specific stochastic aspects of molecular dynamics.

 

Two-dimensional electrophoresis
 

 

The IPGphor II system from Amersham Biosciences

 

Proteome is the protein complement of the genome. Proteomics provides a powerful methodology to investigate protein expression in tissues involved in diseases not linked to particular genetic defects. At present, two-dimensional polyacrylamide gel electrophoresis (2-DE) appears to be the technique of choice for protein expression studies.  In two-dimensional electrophoresis proteins are first separated on a pH gradient on the basis of their isoelectric point (IPGphor II, left), afterwards a standard SDS-PAGE separation takes place discriminating on the basis of the molecular weight (below).

     

Two-dimensional gels may be blotted on PVDF (hydrophobic polyvinylidene difluoride) membrane and incubated with antibodies against proteins of particular interest. Alternatively, the identification of single spots on the 2D gel may be achieved by endoproteinase digestion of excised spots and characterization of the peptide mixture by mass spectroscopy (MS). Furthermore, two-dimensional maps are able to underline post-translational modifications or different levels of protein expression between disease and control tissues eventually linked to different molecular pathways.

 

 

The SE600 and SE900 cells from Hoefer

To this purpose, several network enrichment approaches may be pursuited. We follow a consensus approach by interrogating a number of publicly available tools. Please check Publications for a recent update.