High-performance affinity chromatography complex

In the present paper, we report high-performance affinity chromatography of thrombin in presence of AT III and Hep, using two types of resins as stationary phases either heparin-like PSSO or AT Ill-like PAOM. In order to differentiate their mechanisms of interaction with thrombin, we examined the chromatographic behavior of thrombin in the presence, or in the absence of AT III and/or heparin. Finally, thrombin was injected on the columns at low ionic strength. The desorption of bound thrombin from the two solid surfaces was then carried out using AT III, heparin and the AT Ill-Hep complex, to elucidate the specificity of the interactions involved. 

High-performance affinity chromatography has recently been reported with trypsin-modified avidin supported on 5 pm silica. While the separations were successful and a wide range of foods were studied, elution times were 80 minutes and ADAM post-column reactions were still required (Hayakawa et al. 2009). However, such affinity columns within a solid-phase extraction (SPE) platform make realistic choices for sample preparation, whereby the biotin can be purified and concentrated prior to reversed-phase HPLC. R-Biopharm has recently developed a commercially available antibody-based immunoaffinity column to bind biotin from aqueous extracts, providing an excellent technique to clean up complex samples. 

Affinity separation of proteins in immobilized metal ion affinity chromatography (IMAC). The method is based on the fact that some protein molecules, having specific residues on the surface, can form complexes with metal ions immobilized on the sorbent matrix. Retention is determined by the coordination interactions forming the complexes. Immobilized metal ion affinity chromatography can be performed under very mild, nondenaturing conditions with extremely high selectivity it is particularly suitable for preparative group fractionation of complex extracts and bio-fluids. 

Although an LC/MS combination provides high-resolution separation and the specific detection of analytes in a complex mixture, in many situations it is necessary to provide purification, concentration, and isolation of desired components (e.g., in a biological matrix). This step is performed by affinity chromatography. Practical examples are the selective capture of a specific antigen by the corresponding antibody and the isolation of phosphorylated proteins and peptides [96,97]. An... 

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