Purification and identification of ABA-binding proteins and antibody preparation

✍ Scribed by Yinsheng Wan; Karl H. Hasenstein

• Publisher: John Wiley and Sons
• Year: 1996
• Tongue: English
• Weight: 575 KB
• Volume: 9
• Category: Article
• ISSN: 0952-3499
• DOI: 10.1002/(sici)1099-1352(199634/12)9:5/6<722::aid-jmr329>3.0.co;2-x

✦ Synopsis

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 4 M urea or M ABA. ABA could elute the binding proteins but other phytohormones, such as M A or GAS, could not. ABA binding activity was detected in ABA-and urea-elute fractions using competitive ELISA block tests and [%]ABA binding assays. Scatchard analysis showed an apparent Kd 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 a-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.