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Identification and characterization of 18kDa aleurone specific protein in red rice (Oryza sativa L.)

Author(s): Muthukumar M, Sandeep Kumar, Umesh Hudedamani and KN Singh

Journal: GERF Bulletin of Biosciences
ISSN 2229-6433

Volume: 3;
Issue: 2;
Start page: 9;
Date: 2012;
Original page

Keywords: Aleurone specific protein | 18kDa | red rice | RSP-antibody | immune-staining techniques

An aleurone specific protein of 18kDa, localized in the outermost aleurone layer of red rice seed was isolated, purified and characterized using immune-staining techniques. Aleurone proteins were eluted by reverse phase-high performance liquid chromatography (RP-HPLC) with lesser retention time than white endosperm and total proteins. This analysis also confirmed the hydrophilic nature of this protein. Profile profiling of total soluble, endosperm and aleurone proteins on a 15% Sodium dodecyl sulphate- polyacrylamide (SDS-PAGE) gel showed a distinct band representing a polypeptide of 18 kDa identified in the total and aleurone protein samples but not in the white endosperm. This protein was presumed to be aleurone specific and polyclonal antibodies raised against the aleurone proteins in rabbit was designated as rice seed protein-antibody (RSP-antibody). The antibody titre was determined as 1:1000 by indirect enzyme linked immune sorbent assay and antibody specificity to aleurone protein was confirmed by western and dot blot assays using RSP-antibody. The location of identified aleurone protein was proved by tissue print immunolocalization assay using RSP antibody. These immunostaining techniques proved that this protein was unique to the aleurone layer of the rice grain.
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