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Effective protein extraction protocol for proteomics studies of J erusalem artichoke leaves
Author(s) -
Zhang Meide,
Shen Shihua
Publication year - 2013
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.201300199
Subject(s) - trichloroacetic acid , chemistry , acetone , chromatography , protein purification , extraction (chemistry) , fractionation , phenol extraction , ammonium sulfate , acetic acid , biochemistry , rna , gene
Protein extraction is a crucial step for proteomics studies. To establish an effective protein extraction protocol suitable for two‐dimensional electrophoresis (2 DE ) analysis in J erusalem artichoke ( H elianthus tuberosus L .), three different protein extraction methods—trichloroacetic acid/acetone, M g/ NP ‐40, and phenol/ammonium acetate—were evaluated using J erusalem artichoke leaves as source materials. Of the three methods, trichloroacetic acid/acetone yielded the best protein separation pattern and highest number of protein spots in 2 DE analysis. Proteins highly abundant in leaves, such as R ubisco, are typically problematic during leaf 2 DE analysis, however, and this disadvantage was evident using trichloroacetic acid/acetone. To reduce the influence of abundant proteins on the detection of low‐abundance proteins, we optimized the trichloroacetic acid/acetone method by incorporating a PEG fractionation approach. After optimization, 363 additional (36.2%) protein spots were detected on the 2 DE gel. Our results suggest that trichloroacetic acid/acetone method is a better protein extraction technique than M g/ NP ‐40 and phenol/ammonium acetate in J erusalem artichoke leaf 2 DE analysis, and that trichloroacetic acid/acetone method combined with PEG fractionation procedure is the most effective approach for leaf 2 DE analysis of Jerusalem artichoke.