Électrophorèse sur gel de polyacrylamide

Résumé

L'électrophorèse sur gel de polyacrylamide ou PAGE est une technique utilisant un gel réticulé fabriqué au moment de l'emploi en mélangeant de l'acrylamide qui polymérise sous l'action de l'APS (persulfate d'ammonium ; réactif qui initie la réaction) et du TEMED ; catalyseur de la polymérisation] en donnant des chaînes linéaires. Le gel ainsi formé possède un réseau, dont les mailles sont de taille variable en fonction des proportions d'acrylamide et de bis-acrylamide utilisée, le gel obtenu se comporte donc comme un tamis moléculaire. Les molécules biologiques sont alors faites migrées le long du gel sous l'action d'un champ électrique. Plusieurs techniques existent ensuite afin de les révéler. Utilisation Un gel de polyacrylamide est une matrice de séparation utilisée en électrophorèse de biomolécules, telles que les protéines ou les fragments d'ADN. Les techniques traditionnelles de séquençage de l'ADN telles que les méthodes de Maxam-Gilbert ou de Sanger utilisent les

Source officielle

https://fr.wikipedia.org/wiki/%C3%89lectrophor%C3%A8se_sur_gel_de_polyacrylamide

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New analytical tools combining gel electrophoresis and mass spectrometry

Elena Tobolkina

Proteomics has been one of the main projects challenging biological and analytical chemists for many years. The separation, identification and quantification of all the proteins expressed within biological systems remain the main objectives of proteomics. Due to sample complexity, the development of fractionation, separation, purification and detection techniques that possess appropriate resolution to separate a large number of proteins, as well as being sensitive and fast enough for high throughput protein analysis are required. The objective of this thesis was to develop new separation strategies for protein/peptide fractionation using gel electrophoresis and its further detection by mass spectrometry analysis. A multi-electrode set up based on OFFGEL electrophoresis was developed. The objective was to provide a more efficient application of the electric field for fast and improved protein separation. The results showed that using a multi-electrode setup provides not only a higher protein separation but also better protein collection efficiency. Electrophoretic separation using an ultra narrow pH gradient (UNPG) gel was adapted for a three-well OFFGEL device for fast sample purification and desalting. Purification of an E.coli extract was applied to demonstrate that electrophoretic separation with UNPG gels provides an efficient strategy for fast purification of complex biological samples and can be utilized as a preparative technique in proteomics. UNPG gels were also used to separate charge molecules taking place in a new electro-elution device. The molecules were washed from the gel surface by an aqueous buffer and collected for further analysis by mass spectrometry. The electroelution device provides a fast approach that avoids time-consuming steps of extraction from the polyacrylamide gel. An electrostatic spray ionization (ESTASI) mass spectrometry technique was developed in our group to ionize sample solutions on different substrates. ESTASI has been here coupled to isoelectric focusing and demonstrated to be a powerful tool, which can improve the detection sensitivity compared to standard visualizing protocols. Paper-ESTASI-MS was developed and applied for rapid perfume analysis of six authentic fragrances as a high throughput and sample preparation-free method. ESTASI was also applied for quantitative analysis of caffeine in different beverages using a standard addition strip. The results were compared to classical standard addition methods for MS or LC. It was shown that the strip strategy could be utilized for fast and accurate food analysis and quality control. Desalting and direct analysis of samples from ZipTip by ESTASI-MS has been demonstrated.

EPFL2014

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Cell therapy could be a solution for many diseases that currently pose a huge health burden on patients. Especially, cell therapies based on cell microencapsulation are of great interest. Immunoprotection of cells would allow to avoid the need for immunosuppression and the need for human donors, as the use of xenogeneic cell sources could be feasible. An optimal biomaterial for the microencapsulation of cells must be tailored for the type of cells and the intended application. Hydrogels are great candidates for cell immobilization, due to their high biocompatibility and extracellular matrix-like properties. Amongst them, alginate has outstanding potential with its favorable gelling properties, high biocompatibility, and relatively easy availability. However, in many aspects, it cannot fulfill the requirements of an optimal biomaterial for cell immobilization and transplantation. It lacks good mechanical properties, long-term durability and stability, and has poor shape recovery ability. In addition, severe immune reactions of the host system to the transplanted microcapsules can happen, which eventually result in cell death and early graft failure. Hence, the main focus of the present project was functionalization of alginate to develop new hydrogel materials with improved properties for cell microencapsulation. First, the thesis presents a robust, straightforward strategy and its optimization for the functionalization of alginate on the hydroxyl groups with poly(ethylene)glycol (PEG) via stable carbamate bond. The end-functionality of the heterobifunctional PEG provides a reactive group for covalent crosslinking. Hence, dual ionic-covalent microsphere preparation is achieved by extrusion of the aqueous polymer solution into a gelation bath containing ionic cross-linker and followed by concomitant covalent cross-linking. The possibility of using a variety of covalent cross-linkers was explored. Depending on the end-functionality of the heterobifunctional PEG, the chemical structure of the cross-linker is adjustable. This thesis presents thiol-thiol, thiol-acrylate, thiol-maleimide and thiol-acrylamide interactions, and compares the mechanical properties of the resulted microspheres to those of Ca-alginate. Both the mechanical resistance and the shape recovery performance of microspheres stabilized by an additional chemical cross-linking was improved. In addition, the stability of these microspheres towards non gellifying solutions was significantly enhanced. Still, biomaterials are exposed to the risk of immune responses when transplanted, such as inflammation and fibrosis. Introduction of the anti-inflammatory drug ketoprofen to alginate backbone through a PEG linker and consecutive microsphere formation provides controlled ketoprofen release. Based on this strategy, development of new types of anti-fibrotic derivatives of pirfenidone were realized. Conjugation of the most potential derivative to PEG followed by grafting on alginate resulted in a polymer capable of forming microspheres. Covalent conjugation of the drug was further compared to co-encapsulation of the drug with Ca-alginate. Eventually, the thesis presents the development of multicomponent hybrid hydrogel microspheres, combining the introduction of covalent cross-linking and anti-inflammatory compounds within the hydrogel network. The feasibility of using the newly developed biomaterials are confirmed by in vitro and in vivo assays.

EPFL2020

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Characterization of the electron-accepting part of the organohalide respiration chain of Dehalobacter and Desulfitobacterium

Lorenzo Cimmino

Organohalide respiration (OHR) is a bacterial anaerobic process that use halogenated compounds, e.g. tetrachloroethene (PCE), as terminal electron acceptors. D. restrictus strain PER-K23, an obligate OHR bacterium (OHRB), and D. hafniense strain TCE1, a bacterium with a versatile metabolism, harbour the pceABCT gene cluster, which represents our model system for the study of PCE respiration. To date, the function of PceA, the key enzyme in the process, and PceT, the molecular chaperone for PceA maturation, are well defined. However, the role of PceB and PceC are still not elucidated and the biochemistry of OHR electron transfer is still relatively elusive. Based on the genetic composition of the pce gene cluster, the hypothesis that PceB and PceC may play a role in electron transfer in the metabolism of organohalide respiration is tempting but the question remains largely unanswered. In the present Ph.D. thesis, a multilevel study aiming at characterizing the electron-accepting part of the respiratory chain of OHRB will be presented. The investigation was assessed at molecular level by the deciphering of the stoichiometry of pceABCT individual gene products, and at physiological and biochemical levels, where the characterization of the membrane PceA-containing protein complex of both obligate and facultative OHRB was the main focus. The stoichiometry analysis at RNA level revealed that the pce gene cluster is an operon with, however, a level of transcription that differs for individual genes, an observation that could be explained by post-transcriptional events. At proteomic level, an apparent 2:1 stoichiometry of PceA and PceB was obtained in the membrane fraction, while low abundance of PceC in comparison to the other two proteins was observed. In the soluble fraction, a 1:1 stoichiometry of PceA and PceT was identified. Furthermore, a combination of Clear-Native PAGE gel and a in-gel PceA enzymatic activity assay were applied to identify the RDH complex from the membrane of D. restrictus and D. hafniense strain TCE1. The results revealed an active RDH complex in the membrane extract of both organisms with an estimated molecular mass of 180 kDa, while no RDH activity could be detected in the soluble fraction. Furthermore, immunoblotting for PceA on CN-PAGE gel revealed the presence of a second, however inactive, PceA-containing complex with a molecular mass of 670 kDa, which was confirmed by MS analysis to be largely dominated by the molecular chaperone GroEL alongside with PceA and likely representing the GroEL maturation complex. The RDH complex was purified from the membrane extract by chromatography. The obtained fractions were analysed by LC-MS/MS showing unambiguously the presence of PceA and PceB. Furthermore, preliminary cryo-EM analysis led to propose a 3D reconstruction of the RDH complex, revealing the likely presence of a Pce(AB)2 complex. In summary, this study represents a road-map for the identification of RDH complexes from OHRB. The identification of PceA associated with the GroEL maturation complex raised new questions about the biosynthetic pathway of PceA and invites to consider the involvement of GroEL into the maturation of the key enzyme in organohalide respiration. Finally the preliminary results obtained via cryo-EM analysis set the basis for further investigation on the structure of the RDH complex and open new horizons towards the elucidation of the electron transfer chain involved in the reduction of PCE.

EPFL2022

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Characterization of the electron-accepting part of the organohalide respiration chain of Dehalobacter and Desulfitobacterium

Lorenzo Cimmino

EPFL2022

New analytical tools combining gel electrophoresis and mass spectrometry

Elena Tobolkina

EPFL2014

EPFL2020