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Zinc Blotting Assay for Detection of Zinc-Binding Prolamin in Barley (Hordeum vulgare) Grain

¹Department of Molecular Biology and Genetics, Faculty of Science and Technology, Aarhus University, Forsøgsvej 1, DK-4200 Slagelse, Denmark. ²Corresponding author. Phone: +45 871 58242. Fax: +45 871 56072. E-mail: eva.vincze@agrsci.dk

In plants, zinc is commonly found bound to proteins. In barley (Hordeum vulgare), major storage proteins are alcohol-soluble prolamins known as hordeins, and some of them have the potential to bind or store zinc. ⁶⁵Zn overlay and blotting techniques have been widely used for detecting zinc-binding protein. However, to our knowledge so far this zinc blotting assay has never been applied to detect a prolamin fraction in barley grains. A radioactive zinc (⁶⁵ZnCl₂) blotting technique was optimized to detect zinc-binding prolamins, followed by development of an easy-to-follow nonradioactive colorimetric zinc blotting method with a zinc-sensing dye, dithizone. Hordeins were extracted from mature barley grain, separated by SDS-PAGE, blotted on a membrane, renatured, overlaid, and probed with zinc; subsequently, zinc-binding specificity of certain proteins was detected either by autoradiography or color formation. The dithizone staining method gave similar reproducibility to the radioactive blotting. The detected zinc-binding protein was identified as B-hordein by Western blotting.