Purification methods affinity chromatography

Efficient purification by affinity chromatography depends on the nature of the ligand, the methods used in the preparation of the chromatographic packing,... 

To allow all culture productiou to be coutrolled, a method for rapid analysis is required. Prior to development of an LC-MS method, the analysis was both complex and time-consuming, involving the purification of a relatively large amount of the antibody using affinity chromatography, enzymatic release, and subsequent derivatizafion of the oligosaccharides and their analysis by using capillary electrophoresis. 

The affinity chromatography on ConA - cellulose indicated the presence of small N-glycosylation of all forms of exopolygalacturonases present in carrot roots (unpublished results). This method was usefull for purification of these enzymes from other protein inpurities but was completely uneffective by separation of individual forms (Fig. 4). 

The endothelin B receptor is an example of characterization of a homogeneous, affinity purified protein (Roos et al., 1998). Significant progress has been made in the development of techniques for more high-throughput identification of phosphorlyation events. Analysis of large sets of phosphorylated proteins is facilitated by the availability of affinity purification methods such as anti-phosphotyrosine or anti-phosphoserine antibodies or metal affinity chromatography (Neubauer and Mann, 1999 Soskic et al., 1999). These methods are not specific to a particular protein but rather are used to fractionate all proteins that are phosphorylated. 

In this chapter, we describe our methods for purification of eIF2B holo-complexes as well as the catalytic subcomplex and the catalytic eIF2Bfi subunit (encoded by GCD6 in yeast) alone. We typically epitope tag only one eIF2B subunit, over-express the desired subunit combination, and use a single affinity chromatography step to purify the desired complex. In early work, a... 

Ruiz-Carrillo, A., and Allfrey, V.G. (1973) A method for the purification of histone fraction F3 by affinity chromatography. Arch. Biochem. Biophys. 154, 185-191. 

This method involves passing the protein through a column filled with resins of unique characteristics. Depending on the type of the resin or beads, purification can be achieved through (i) Ion Exchange, (ii) Size Exclusion or (iii) Affinity Chromatography. 

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