Abstract:
:The presence of high-abundance proteins in complex protein mixtures often masks low-abundance proteins and causes loss of resolution of 2DE. Protein fractionation steps conducted prior to 2DE can enhance the detection of low-abundance proteins and improve the resolution of 2DE. 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 Escherichia 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 2DE, 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 2DE.
journal_name
Proteomicsjournal_title
Proteomicsauthors
Wang W,Wu X,Xiong E,Tai Fdoi
10.1002/pmic.201100475subject
Has Abstractpub_date
2012-04-01 00:00:00pages
938-43issue
7eissn
1615-9853issn
1615-9861journal_volume
12pub_type
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