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Improving gel‐based proteome analysis of soluble protein extracts by heat prefractionation
Author(s) -
Wang Wei,
Wu Xiaolin,
Xiong Erhui,
Tai Fuju
Publication year - 2012
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.201100475
Subject(s) - proteome , fractionation , chemistry , chromatography , protein purification , reagent , biochemistry , denaturation (fissile materials) , proteomics , gene , nuclear chemistry
The presence of high‐abundance proteins in complex protein mixtures often masks low‐abundance proteins and causes loss of resolution of 2 DE . Protein fractionation steps conducted prior to 2 DE can enhance the detection of low‐abundance proteins and improve the resolution of 2 DE . Here, we report a method to prefractionate soluble protein extracts based on protein thermal denaturation. Soluble proteins were extracted from maize embryos and leaves and E scherichia coli cells. Through heating at 95° C for 5 min, soluble protein extracts were prefractionated as heat stable protein fraction (the supernatant) and heat labile protein fraction (the precipitate). Our results showed that heat prefractionation enhanced the separation of proteins in both fractions by 2 DE , thereby increasing the chance of detecting low‐abundance proteins, many of which were nonvisible in unfractionated extract. In maize embryo, 330 spots were detected in soluble protein extract, while 577 spots were detected after prefractionation. Furthermore, this prefractionation method facilitated the enrichment, detection, and identification of de novo synthesized stress proteins. Because of its simplicity, the one‐step heat prefractionation minimizes protein loss. Finally, heat prefractionation requires no expensive special hardware or reagents, and provides an alternative prefractionation for increasing the resolving power of 2 DE .