Besides connecting biotin to a chemical group that mediates protein attachment, spacer arms can influence biotinylation and protein detection in three ways. Spacer arms link the biotin molecule to a reactive group that interacts with certain functional groups on the amino acids of the target protein. Variations in these three features account for the many varieties of available reagents and provide the specific properties needed for particular applications. They are composed of the biotinyl group, a spacer arm and a reactive group that is responsible for attachment to target functional groups on proteins. Chemical methods of biotinylation are most commonly used, and the biotinylation reagents used for this type of labeling share several basic features. In those situations, certain variants of avidin or derivatives of biotin are available, which allow soft-release (elution) binding or cleavable (reversible) labeling.īiotinylation is the process of labeling proteins or nucleotides with biotin molecules and can be performed by enzymatic and chemical means. However, depending on the nature of the application, the very strong binding interaction can be problematic. Together, these features make avidin–biotin strategies ideal for many detection and immobilization applications. Additionally, biotin (244.3 Da) is considerably smaller than enzyme labels and is therefore less likely to interfere with normal protein function. Indeed, this interaction is one of the strongest noncovalent interactions between a protein and ligand. By coupling mathematical relationships describing these elution mechanisms to the solution of the continuity equation, protein elution times are accurately predicted.Biotin is a useful label for protein detection, purification and immobilization because of its extraordinarily strong binding to avidin, streptavidin or Thermo Scientific NeutrAvidin Protein. The model predicts how simultaneous modulation of ionic strength and pH during elution can greatly improve the separation of proteins with similar pI’s elution characteristics including retention time, peak width and resolution can likewise be improved. Gradient sculpting methods are used to enhance ICF as the first dimension in a multidimensional separation platform used for the detection and analysis of O-linked N-acetylglucosamine modified proteins within the proteome of differentiated C2C12 mouse myoblast cells.įinally, a model of protein transport and binding in ICF is developed and used to show that elution is not dictated solely by a protein’s isoelectric point (pI), but is instead multi-modal in nature with Donnan equilibria, ion-exchange, and ion-displacement effects at work. The method is used to achieve high purity and process throughput of a desired isoform of recombinant N-lobe of human transferrin produced by Pichia pastoris using custom isocratic ICF on preparative media. The model can be used for in silico design of custom-shaped elution profiles to improve separation performance. The buffers used are selected to satisfy cost constraints and for compatibility with detection by UV absorption at 280 nm and mass spectrometry.Ī new surface-reaction/chemical-equilibria model is derived and solved by computer-aided simulations to predict pH and ionic strength profiles generated on anion and cation exchange columns. On anion and cation exchange media, linear gradients spanning more than six pH units are generated through isocratic or gradient interchange of loading and elution phases. Work presented in this thesis focuses on advancing ICF at both analytical and preparative scales.Ī method for generating pH gradients in ICF is developed using simple low-molecular-weight buffers. This is partly due to poor knowledge about operating ICF at scale, lack of understanding of its elution mechanisms, and the use of complex, costly buffers.
Isoelectric chromatofocusing (ICF), a mode of chromatography by which proteins are separated based on changes in their charge with pH, is widely used at analytical scales, but its use in bio-product manufacturing has been limited.
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