Abstract
Poor detection of low-abundant proteins is a common problem in two-dimensional electrophoresis (2-DE) for separation of proteins in a proteome analysis. This is attributed partially, at least, to the existence of high-abundant proteins, e.g. ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in plants. They engage a large proportion of the whole-cell proteins and thus prevent low-abundant proteins from being up-taken by immobilized pH gradient (IPG) strip, consequently making the latter poorly detectable by 2-DE. In this work, we report a straightforward protocol for preparation of whole-cell proteins through differential polyethylene glycol (PEG) precipitation aiming at elimination of Rubisco from plant protein samples. In comparison with 2-DE analysis of protein samples prepared using a conventional TCA/acetone method, a relatively high reproducibility of proteins was achieved using a PEG fractionation protocol in terms of protein yield and protein species. As expected, the large subunit of Rubisco was precipitated predominantly in the 16% PEG fraction. This allowed proteins of the Rubisco-containing fraction to be analyzed separately from those of other PEG fractions. After taking into account the overlapping protein spots among 2-DE gels of all fractions through image and statistical analyses, we detected with this protocol a total 5077 protein spots, among which ca. 80% are proteins undetectable with the TCA/acetone method, while the rest of proteins exhibited a significant increase in their abundance. This protocol was developed using Arabidopsis as a source of protein and thus may also be applicable to protein preparations of other plants.
Original language | English (US) |
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Pages (from-to) | 2341-2348 |
Number of pages | 8 |
Journal | Phytochemistry |
Volume | 67 |
Issue number | 21 |
DOIs | |
State | Published - Nov 2006 |
Externally published | Yes |
Keywords
- Arabidopsis thaliana
- Cruciferae
- Low-abundant proteins
- Polyethylene glycol
- Protein preparation
- Rubisco
- Two-dimensional electrophoresis
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Plant Science
- Horticulture