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Browsing by Author "Battistuzzi, Cristina"

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  • Battistuzzi, Cristina (2019)
    The large variety of shapes, functions, and conformations of proteins explains the challenges in protein identification and separation in solution. Gel electrophoresis, and more specifically SDS-PAGE, is an established technique applied to large proteins. Capillary gel electrophoresis offers the advantage of miniaturization coupled with higher speed of analysis and sample throughput. Protein analysis in silica capillaries is affected by the presence of charges on the silica surface, causing absorption, reliability and repeatability issues. Electroosmotic flow may also contribute, requiring buffer additives such as sodium dodecyl sulphate and dynamic or permanent coatings on the capillary wall. Coatings employed in capillary zone electrophoresis can be neutral or charged. They include copolymers such as poly((1-vinylpyrrolidone)-co-(2-dimethylaminoethyl methacrylate)), polyacrylamide, diazoresin as a coupling agent to form covalently bound coatings with polyvinyl alcohol, carboxyl fullerene and graphene oxide, and proprietary coatings. Capillary gel electrophoresis offers the advantages of a sieving matrix to enhance the separation of large proteins with similar charge-to-mass ratio. Dilute and semi-dilute polymer solutions with or without self-coating properties can be employed, such as polyacrylamide, polydimethyl acrylamide, and hydrophilic cellulose derivatives such as hydroxypropyl cellulose and hydroxyethyl cellulose. UV detection is enhanced by stacking techniques such as field-amplified sample stacking and the addition of sodium chloride to the sample. The focus of experimental laboratory works for this thesis was the identification of BSA, the lipoproteins HDL and LDL, and an apolipoprotein ApoB-100 using capillary gel electrophoresis. The polymer solution employed was cross-linked polyacrylamide. Instrumental factors such as run voltage, injection type, presence of sieving matrix and SDS in the BGE, sample concentration and presence of NaCl in the sample were examined. Repeatability was an issue throughout the study caused by current instability, although SDS in the BGE and addition of NaCl to the sample prior to injection had a positive effect. Stability of the BGE and the sieving matrix, together with addition of NaCl to the sample could be explored further.