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

In plants Zn is commonly found bound to proteins. In barley (Hordeum vulgare) major storage proteins are alcohol soluble prolamin known as hordein and some of them have potentiality to bind/store Zn. 65Zn overlay and blotting techniques have been widely used for detecting zinc binding protein. However, so far of our knowledge this Zn blotting assay was never been applied to detect 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 non-radioactive colorimetric zinc blotting method using a zinc sensing dye, dithizone (DTZ). Hordeins were extracted from mature barley grain, separated by SDS-PAGE, blotted on membrane, renatured, overlayed and probed with zinc and subsequently zinc binding specificity of certain proteins was detected either by autoradiography or colour formation. The DTZ staining method gave similar reproducibility like the radioactive blotting. The detected zinc binding protein was identified as B-hordein by western blotting experiment. 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