Quantitative Analysis of Mitochondrial Protein Expression in Methylmalonic Acidemia by Two-Dimensional Difference Gel Electrophoresis

2006 ◽  
Vol 5 (7) ◽  
pp. 1602-1610 ◽  
Author(s):  
Eva Richard ◽  
Lucia Monteoliva ◽  
Silvia Juarez ◽  
Belén Pérez ◽  
Lourdes R. Desviat ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
Protein Expression ◽  
Gel Electrophoresis ◽  
Mitochondrial Protein ◽  
Methylmalonic Acidemia ◽  
Two Dimensional ◽  
Difference Gel Electrophoresis

scholarly journals Fluorescence two-dimensional difference gel electrophoresis for biomaterial applications

2009 ◽  
Vol 7 (suppl_1) ◽  
Author(s):  
Laura E. McNamara ◽  
Matthew J. Dalby ◽  
Mathis O. Riehle ◽  
Richard Burchmore
Keyword(s):  
Protein Expression ◽  
Gel Electrophoresis ◽  
Two Dimensional ◽  
Expression Levels ◽  
Powerful Technique ◽  
Difference Gel Electrophoresis

Fluorescence two-dimensional difference gel electrophoresis (DiGE) is rapidly becoming established as a powerful technique for the characterization of differences in protein expression levels between two or more conditions. In this review, we consider the application of DiGE—both minimal and saturation labelling—to biomaterials research, considering the challenges and rewards of this approach.

62 COMPARATIVE PROTEOMIC ANALYSIS OF LIVER MITOCHONDRIA DERIVED FROM DECEASED NEWBORN CLONED CALVES AND ADULT CLONES BY TWO-DIMENSIONAL DIFFERENTIAL GEL ELECTROPHORESIS

2011 ◽  
Vol 23 (1) ◽  
pp. 137
Author(s):  
K. Takeda ◽  
M. Tasai ◽  
S. Akagi ◽  
S. Watanabe ◽  
M. Oe ◽  
...  
Keyword(s):  
Protein Expression ◽  
Somatic Cell ◽  
Proteomic Analysis ◽  
Gel Electrophoresis ◽  
Mitochondrial Protein ◽  
Two Dimensional ◽  
Cell Nuclei ◽  
Comparative Proteomic Analysis ◽  
Mitochondrial Fractions

Aberrant reprogramming of donor somatic cell nuclei may result in many severe problems in animal cloning. The inability to establish functional interactions between donor nucleus and recipient mitochondria is also likely responsible for developmental deficiency. However, an understanding of the expressed proteins in cattle is lacking. In the present study, alterations in mitochondrial protein levels between somatic cell nuclear transferred (SCNT) and control animals (mostly produced by AI) were investigated. Nuclear transfer was performed using donor cells prepared from cumulus cells (B1), ear skin, or skeletal muscle from adult Japanese Black cattle, and enucleated in vitro matured oocytes (Holstein or Japanese Black) as previously reported (Akagi et al. 2003). Liver samples were collected from postmortem SCNT calves (CB1-3; 0, 1, and 9 days postnatally) and adult SCNT cattle (CA1-4; 6, 6, 6, and 5 years of age) produced from the same cell line (B1) and preserved at –80°C. Mitochondrial fractions were prepared from the frozen–thawed liver samples by mechanical homogenization and differential centrifugation, and subjected to two-dimensional difference in gel electrophoresis (2D-DIGE) using CyDye™ dyes (Cy2, Cy3, Cy5; GE Healthcare) for specific labelling. Protein expression changes were confirmed by ImageMaster 2D Platinum software with a volume ratio greater than 2.0 (Student’s t-test; P < 0.05). The expression of 5 proteins were up-regulated in SCNT calves compared to control calves (n = 6; Day 250 fetus, 0, 4, 8, 8, and 8 days after birth; P < 0.05). Expressed protein patterning compared to control groups was different among SCNT calves. The protein spots of CB-1 showed great differences compared with other SCNT calves; 13 spots were up-regulated, and 18 spots were down-regulated. In adult SCNT cattle, the concentrations of 3 proteins were higher when compared to control cattle (n = 4; 2, 2, 6, and 8 years of age; P < 0.05). Protein expression was different among individual SCNT animals even if they were produced from the same donor cell source. For example, 9 spots were up-regulated and 7 spots were down-regulated in CA-1. In contrast, no differences were detected in 2 of the SCNT cattle (CA-3 and 4; P < 0.05). Novel proteins were not identified in any of the SCNT cattle or calves. In conclusion, alteration of mitochondrial protein expression levels were observed in non-viable neonatal SCNT calves and varied among SCNT individuals; suggesting that mitochondrial related gene expression may be implicated in early losses. Comparative proteomic analysis represents an important tool for further studies on SCNT animals. We thank Dr. C. A. Pinkert (Auburn Univ.) and Dr. Somfai (NARO) for their assistance. This work was supported by a grant from the NARO, Japan.

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