Immobilized ligand method

Nonspecific protein binding to the solid phase complicates the method and is a selective pressure driving its evolution. The adaptive response has been the development of intrinsically comparative methods in which specific binding to an immobilized ligand is blocked in one out of two otherwise identical samples. When the respective protein components of the samples are compared, specifically bound proteins are present in one but severely depleted in the other. To allow relative quantitation, the two samples can be made isotopically distinct by a chemical or metabolic process and then mixed for an analytical step that avoids intersample variability [15]. 

FIGURE 13.4 The effects of (a) multisite attachment, (b) random or improper orientation, and (c) steric hindrance on an immobilized ligand. (Reproduced from Kim, H.S. and Hage, D.S., Immobilization methods for affinity chromatography, in Handbook of Affinity Chromatography, 2nd edn., Hage, D.S. (ed.), CRC Press, Boca Raton, FL, 2005, Chap. 3.)... 

For rapid confirmation of the molecular mass of a recovered peptide following RPC purification, either ESI-MS or MALDI-MS procedures are now indispensable. Studies on the interaction of peptides with solvent molecules, ions, and free or immobilized ligands in chromatographic or electrophoretic environments have, however, been largely confined to methods that examine global properties of different peptides in the bulk mobile phase. In... 

The immobilized ligand is an essential factor that determines the success of an affinity chromatographic method [12]. The method which is used for affinity ligand immobilization is important because actual or apparent activity of the final column can be affected. Decrease in... 

Removal of metals by immobilized ligands frequently involves a choice of two techniques, a batch method or column chromatography. The batch method can be faster when samples are taken from the supernatant for replicate analyses. This method also can establish when equilibrium (or stasis) has been established. One flaw is that the sample of solid may not be... 

The methods for production of such immobilized ligands and for carrying out affinity purification of IgG are essentially similar whatever ligand is used. Sepharose 4B is probably the most widely used matrix for affinity chromatography, but there are other materials available. Activation of Sepharose 4B is usually carried out by reaction with CNBr this can be carried out in the laboratory before coupling or ready-activated lyophihzed Sepharose can be purchased. The commercial product is obviously more convenient than homemade activated Sepharose, but it is more expensive and may be less active. 

Immobilized ligands prepared by the divinylsulfone method are unstable above pH 8.0. 

The modified mix-and-splif combinatorial method is used for the synthesis of the ligand library yields n<->n members, where n represents the number of different amines chosen. Usually 5 g of each immobilized ligand are synthesized however, this amount depends on the screening strategy preferred (for example, the FITC-based system or the ELISA on beads require less resin than the conventional affinity chromatography). 

Hence, the overall resistance to the mass transfer predicted by this variant of the BAMcomb model is the sum of the resistance in the liquid film and the resistance in the pore fluid. The value of the liquid film mass transfer coefficient Kf can be calculated from literature correlations,400,407,408 while the method for the estimation of the apparent pore liquid mass transfer coefficient Kp can be derived from Eq. (163). The surface interaction between the protein and the immobilized ligand at the internal particle surface can be treated in the same way as for nonporous particles, i.e.,... 

Figure 11 Schematic representation of techniques used for solubilized and immobilized affinity ligands in polyacrylamide gels. (A) Solubilized ligand method the ligand can move freely in the gel-buffer system. (B) Macroligand method the solution of acrylamide contains the macroligand that becomes entrapped within the gel matrix after polymerization. (C) Chemically bound ligand direct copolymerization of polyacrylamide gel with the copolymerizable derivative of the ligand. (Reproduced with permission from Ref. 13.)...

The methods of attachment to the solid support can be divided into two broad categories covalent and noncovalent. Several factors should be considered. First, the ligands should be stable to immobilization, storage, and assay conditions. Second, the immobilized ligands should be accessible to the receptors, which are usually in solution. Third, the method should be efficient. 

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