Biology Reference
In-Depth Information
Chapter 17
2D DIGE for the Analysis of RAMOS Cells Subproteomes
Marisol Fernández and Juan Pablo Albar
Abstract
Overexpression of human polm in a Burkitt's lymphoma-derived B cell line (RAMOS), in which somatic
hypermutation (SHM) is constitutive, induced an increase in somatic mutations in the parental cell line
(Nucleic Acids Res 32:5861-5873, 2004). To further study Polm implications in SHM, a dominant-
negative (DN) mutant of Polm (Polm-DN) was generated which showed moderated overexpression of the
Polm-DN protein. The subcellular prefractionation was used to improve the detection of low-abundance
proteins contained in membrane/organelles and nuclei, which are effi ciently separated from high-abundance
proteins commonly found in the cytosol that might otherwise hamper analysis. Two-dimensional (2D)
difference gel electrophoresis (DIGE) is a technique for comparative proteomics, which improves the
reproducibility and reliability of differential protein expression analysis between samples. The standard
sample included in every gel (Cy2) comprises equal amounts of each sample to be compared, and thus
improves the accuracy of protein quantifi cation between samples from different gels, allowing accurate
detection of small differences in protein levels. The combination of this techniques allowed the detection
in Fraction F2 (membrane/organelles) of 2,111 spots, 55 of them with signifi cant variation (19 increased
and 36 decreased in a ratio >2.0 or <−2.0), and in Fraction F3 (nuclear) of 2,416 spots, 80 of them with
signifi cant variation (51 increased and 29 decreased in a ratio >1.5 or <−1.5).
Key words:
Subcellular proteome, Difference analysis, Protein quantitation, DIGE, Gel-to-gel matching
1. Introduction
The separation of complex protein mixtures is very important in
proteomics to allow the detection of low-abundance proteins for
quantitative and qualitative analysis (
1, 2
). The success in such
analysis depends on standardized and reproducible sample prepa-
ration methods prior to protein separation by 2D gel electropho-
resis (
3, 4
). Due to the complexity of eukaryotic proteomes, it is
convenient to partial fractionate proteomes of a given organism in
order to maximize the coverage of the sample and to increase the
visualization of low-abundance proteins and at the same time make
them accessible for subsequent analysis (
5
). Since both membrane
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