Methods of biochemical analysis (Meth Biochem Anal )
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|Other titles||Methods of biochemical analysis|
|Document type||Journal / Magazine / Newspaper|
Publications in this journal
- Methods of biochemical analysis 01/2011; 54:165-81.
- Methods of biochemical analysis 01/2011; 54:183-201.
- Methods of biochemical analysis 01/2011; 54:269-78.
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ABSTRACT: The above discussion provides examples of how to utilize the possibilities arising from different scenarios, related to the level of information available, to identify low molecular weight organic molecule affinity ligands to target proteins. In Table 10.1 the different published results are summarized in terms of the structure of the ligand, the target protein, and a reference to the relevant publication. Common to all reported cases of small molecule affinity ligands is a considerably lower selectivity and affinity compared to natural protein ligands. This lower affinity has to be compensated with more thorough work in the optimization of binding and elution conditions to obtain significant recoveries and purification factors.Methods of biochemical analysis 01/2011; 54:259-67.
- Methods of biochemical analysis 01/2011; 54:319-38.
Article: Electrophoresis in gels.Methods of biochemical analysis 01/2011; 54:365-77.
Article: Ion exchange chromatography.Methods of biochemical analysis 01/2011; 54:93-133.
- Methods of biochemical analysis 01/2011; 54:451-85.
Article: Purification of pegylated proteins.[Show abstract] [Hide abstract]
ABSTRACT: Separation of PEGylated proteins is challenging because PEG itself is a relatively inert, neutral, hydrophilic polymer and the starting point for PEGylation is a pure protein. Thus, other than molecular weight and size, differences in the physicochemical properties typically used to fractionate proteins may be slight between different PEGylated forms of a protein. The usual properties of electrostatic charge and molecular weight (size) form the basis of the most commonly used separation techniques, particularly IEC, SEC, and ultrafiltration. The main effect of PEGylation on ion-exchange separations is to shield the electrostatic charges on the protein surface and to reduce the strength of interactions with higher PEG chain molecular weight or higher PEGylation extent. Thus, ion exchange can be used very effectively to separate on the basis of PEGylation extent for low extents, but as N increases, the effectiveness of separation rapidly diminishes. Separation of positional isomers is possible by RPC or ion exchange at analytical scale, but it is problematic at the preparative scale due to the small size of the differences in electrostatic interactions between isomers. PEGylation imparts significant changes in molecular weight with each chain added to a protein and there are corresponding increases in molecular size, so SEC and ultrafiltration (and dialysis) are effective methods for separating native and PEGylated proteins. However, the relative size difference between variants differing in PEGylation extent by one adduct reduces with N, so that efficient SEC separation between PEGylated species differing by one PEG chain is not likely to be economic at the preparative scale for N > 3. This holds true even for PEG proteins produced with large PEG polymers (Mr > or =20 kDa). For small PEG polymers (Mr = 2 kDa), only native and PEGylated species can be separated effectively. At the analytical scale, with proper calibration, SEC can provide valuable information on PEGylation extent. Membranes can be used to reduce the concentration of smaller molecular weight species by dialysis but cannot fully remove them, and an operational trade-off between purity and yield is required. Gel electrophoresis can confirm PEGylation reactions have proceeded and indicate the relative purity of products, but its use to confirm PEGylation extent requires further research. The main drawback of separations based solely upon molecular size is that they cannot differentiate between positional isomers. Capillary electrophoresis is an exception, quantitatively combining any or all of size, shape, conformational freedom, and small differences in protein surface properties to separate by both PEGylation extent and positional isomerism. Relative hydrophobicity is a useful property for analytical separations using RPC, but HIC, which is used routinely for production-scale purification of proteins, does not appear to be particularly useful for separation of PEGylated species.Methods of biochemical analysis 01/2011; 54:339-62.
- Methods of biochemical analysis 01/2011; 54:411-39.
Article: Covalent chromatography.Methods of biochemical analysis 01/2011; 54:203-19.
- Methods of biochemical analysis 01/2011; 53:vii-ix, 1-445.
Article: Introduction to chromatography.Methods of biochemical analysis 01/2011; 54:25-50.
- Methods of biochemical analysis 01/2011; 54:441-50.
- Methods of biochemical analysis 01/2011; 54:379-409.
- Methods of biochemical analysis 01/2011; 54:489-506.
- Methods of biochemical analysis 01/2011; 54:135-64.
Article: Affinity chromatography.Methods of biochemical analysis 01/2011; 54:221-58.
- Methods of biochemical analysis 01/2011; 54:51-91.
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