Immunoaffinity chromatography protocol

Especially if samples with a high content of unspecific proteins, e.g., sera, are processed by immunoaffinity chromatography, it is recommended to use a sequential washing/elution protocol When all sample is applied, wash the column with PBS or TBS until O.D.280 < 0.1, then with 1 column volume 1 M sodium chloride, followed by 5 vol. PBS or TBS. Elute with 1 vol. elution buffer, e.g., glycine-HCl pH 2.5, then apply 1 vol. alkaline buffer, e.g., 0.1 M borate pH 10. Finally, regenerate the column with PBS or TBS. 

Immunoaffinity chromatography can provide extensive purification of endogenous hormones in plant extracts [60] (see Figs. 6 and 7 in Section 6.2). Both monoclonal and polyclonal antibodies have been used to produce immunoaffinity supports for lAA [60,61], GAs [62,63] and cytokinins [64,65]. Despite the enormous potential of the procedure, it has as yet not found widespread application in plant hormone purification protocols. The situation is unlikely to change until a range of immunoaffinity supports are available from commercial sources at affordable prices. The raising of antibodies against plant hormones, the preparation of a variety of immunoaffinity supports and their application in plant hormone analysis are discussed and evaluated in Chapter 3. 

Each tissue contains not only varying levels of lAA, but also, in far greater quantities, its own characteristic assortment of impurities. It is therefore unsafe to assume that a purification protocol that provides accurate data with one tissue will be similarly effective with extracts from other plant material. This point can be illustrated with data obtained in studies with shoots of Pinus sylvestris and Zea mays [93] in which endogenous lAA in acidic, diethylether extracts was analysed by ion-suppression, reverse phase HPLC with a fluorimetric detector both before and after purification by immunoaffinity chromatography... 

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