Off-Gel[TM] electrophoresis: characterisation of a novel isoelectric focusing fractionation method

The Off-Gel™ technology is an isoelectric focusing (IEF) fractionation method which enables the purification of one or several proteins in a solution placed on an immobilised pH gradient (IPG) gel. The present thesis is devoted to its characterisation and validation, experimentally and with the help of numerical simulation. As the major component of the device, the IPG gel has been studied in terms of pH gradient and conductivity. This chapter provides a first experimental framework for the recently developed Off-Gel™ electrophoresis. The focusing and separation of two ampholytes A and B present in an aqueous solution next to a gel buffered by immobilised acid moieties IH has been studied by finite element simulation in an iterative scheme, with the assumption of the presence of a salt in excess. The model has been firstly formulated in a mono-dimensional domain, taking into account transient diffusion and equilibrium kinetics of the two amphoteric species A and B, of water and of the immobilised species IH. The first calculation has illustrated the pH evolution between an ampholyte solution and an IPG gel, and the influence of the Immobiline concentration on protons and ampholyte distributions. The model has later been completed, in a two-dimensional domain, with the addition of the migration term and the application of a pH gradient in the gel. The final simulation has shown the neutralisation of the ampholyte A in the solution chamber and the migration of B from the chamber to the gel. It is next demonstrated that the Off-Gel™ approach can be used to desalt biological samples (i.e. to remove inorganic salts not compatible with mass spectrometry (MS) analysis). A mathematical model has been developed to estimate the decrease in conductivity of an electrolyte solution under the effect of an applied electric field. A good agreement has been found by comparison with conductivity measurements. By using finite element simulation, the migration of the salt from the aqueous solution to the gel has been characterised. It illustrates how the conservation of electroneutrality and a stacking effect can slowdown the process when the classical assumption of supporting salt in excess is not verified. The Off-Gel™ technology is then validated with the fractionation of model proteins. The proteins are separated according to their isoelectric point with the advantage to be recovered in solution for further analysis. The protein fractions obtained with this technique, working in recirculation mode, have been analysed with MS. A conductometric detection directly implemented in the solution chamber has also been validated. A multi-compartment Off-Gel™ format is next presented with the fractionation of pI protein markers in order to highlight the efficiency of the technique. One species numerical simulation has been developed to visualise the electric field distribution during Off-Gel™ separation in the multi-well system. The resolution of the two devices has been shown to be 0.1 pH unit for the separation of ßlactoglobulin A and B. Further applications carried out with real biological samples has proved the performances of the technique, notably for proteomics. Preliminary results are finally given with the separation of different size gold nanoparticles by Off-Gel™ electrophoresis. A colloidal solution with a broad size distribution has been successfully fractionated via In-Gel electrophoresis. Gold nanoparticles with a narrow size distribution and a standard deviation lower than 5 % have been obtained.

    Thèse École polytechnique fédérale de Lausanne EPFL, n° 3064 (2004)
    Section de chimie et génie chimique
    Faculté des sciences de base
    Institut des sciences et ingénierie chimiques
    Laboratoire d'électrochimie physique et analytique
    Jury: Christos Comninellis, Kai Johnsson, Wolfgang Thormann, Jean-Daniel Tissot

    Public defense: 2004-7-30


    Record created on 2005-03-16, modified on 2016-08-08


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