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Zinc Blotting Assay for Detection of Zinc‐Binding Prolamin in Barley ( Hordeum vulgare ) Grain
Author(s) -
Uddin Mohammad Nasir,
Nielsen Ane LangkildeLauesen,
Vincze Eva
Publication year - 2014
Publication title -
cereal chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.558
H-Index - 100
eISSN - 1943-3638
pISSN - 0009-0352
DOI - 10.1094/cchem-09-13-0175-n
Subject(s) - hordeum vulgare , blot , zinc , chemistry , hordein , dithizone , biochemistry , storage protein , chromatography , biology , botany , poaceae , organic chemistry , gene
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. 65 Zn 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 ( 65 ZnCl 2 ) 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.