Purification and identification of ABA-binding proteins and antibody preparation

Journal of Molecular Recognition : JMR
Y Wan, K H Hasenstein

Abstract

Maize root membranes were extracted and the solubilized proteins were affinity-purified using an ABA-BSA-Sepharose 4B matrix. The retained proteins were eluted with 4M urea or 10(-4)M ABA. ABA could elute the binding proteins but other phytohormones, such as IAA or GA3, could not. ABA binding activity was detected in ABA- and urea-elute fractions using competitive ELISA block tests and [3H]ABA binding assays. Scatchard analysis showed an apparent K(d) of 4.8 nM for the ABA binding activity of the protein. When ABA or urea eluate was loaded on a concanavalin-A-Sepharose column, the fraction eluted with 0.2 M methyl alpha-mannopyranoside still showed ABA binding activity, suggesting that ABA binding proteins are glycoproteins. Polyclonal antibodies against ABA binding proteins were raised using as immunogen ABA or urea eluate from the ABA-BSA-Sepharose column. The resulting antibodies not only recognized 56 kDa binding proteins but also blocked the binding of ABA to an ABA-specific antibody, indicating properties similar to anti-idiotypic antibodies. The purified antibodies will be suitable to purify and characterize putative ABA receptors.

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