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Depletion of abundant plant R u B is CO protein using the protamine sulfate precipitation method
Author(s) -
Kim Yu Ji,
Lee Hye Min,
Wang Yiming,
Wu Jingni,
Kim Sang Gon,
Kang Kyu Young,
Park Ki Hun,
Kim Yong Chul,
Choi In Soo,
Agrawal Ganesh Kumar,
Rakwal Randeep,
Kim Sun Tae
Publication year - 2013
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201200555
Subject(s) - rubisco , protein precipitation , proteomics , chemistry , biochemistry , biology , proteome , western blot , protein purification , botany , chromatography , extraction (chemistry) , photosynthesis , gene
Ribulose‐1,5‐bisphosphate carboxylase/oxygenase ( R u B is CO ) is the most abundant plant leaf protein, hampering deep analysis of the leaf proteome. Here, we describe a novel protamine sulfate precipitation ( PSP ) method for the depletion of R u B is CO . For this purpose, soybean leaf total proteins were extracted using T ris‐ M g/ NP ‐40 extraction buffer. Obtained clear supernatant was subjected to the PSP method, followed by 13% SDS ‐ PAGE analysis of total, PS ‐supernatant and ‐precipitation derived protein samples. In a dose‐dependent experiment, 0.1% w/v PS was found to be sufficient for precipitating R u B is CO large and small subunits ( LSU and SSU ). Western blot analysis confirmed no detection of R u B is CO LSU in the PS ‐supernatant proteins. Application of this method to A rabidopsis , rice, and maize leaf proteins revealed results similar to soybean. Furthermore, 2 DE analyses of PS ‐treated soybean leaf displayed enriched protein profile for the protein sample derived from the PS ‐supernatant than total proteins. Some enriched 2 D spots were subjected to MALDI ‐ TOF ‐ TOF analysis and were successfully assigned for their protein identity. Hence, the PSP method is: (i) simple, fast, economical, and reproducible for R u B is CO precipitation from the plant leaf sample; (ii) applicable to both dicot and monocot plants; and (iii) suitable for downstream proteomics analysis.