2DGETwo-Dimensional Gel Electrophoresis (protein analysis)
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Caption: Figure 3: Two-dimensional gel electrophoresis (2DGE) image analysis of the liver of aged rats following exercise.
The extracted whole cell lysates of both samples were subjected to 2DGE in which more than 1000 protein spots were able to be consistently resolved and detected for both control and treated samples.
In this study, we employed the 2DGE and MALDI-TOF/TOF-MS to identify the differentially expressed proteins in this cell line upon exposure to MNQ at 7.5 [micro]M.
Abbreviations: 2DGE, two-dimensional gel electrophoresis; CHAPS, 3-[(3-cholamidopropyl)dimethylammoniol-1- propanesulfonate; AP-1, activator protein-1; DTT, dithiothreitol; DMSO, dimethyl sulphoxide; EMT, epithelial-to-mesenchymal transition; ERK1/2, extracellular signal-regulated kinase 1/2; FAR, focal adhesion kinase; FBS, fetal bovine serum; IPG, immobilised pH gradient; MALDI-TOF/TOF-MS, matrix-assisted laser desorption ionisation tandem time-of-flight mass spectrometry; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; MNQ, 2-methoxy-1,4-naphthoquinone; NF-[kappa]B, nuclear factor-[kappa]B; NMIIA, nonmuscle myosin-IIA; ROS, reactive oxygen species; RPSA, laminin-binding protein; S100A4, protein S100-A4; uPa, urokinase plasminogen activator,
[5] Nonstandard abbreviations: 2DGE, 2-dimensional gel electrophoresis; ANIT, [alpha]-naphthylisocyanate; MS, mass spectrometry; MCI, master cluster index; MALDI, matrix-assisted laser desorption/ionization; ALT, alanine aminotransferase; PNP, purine nucleoside phosphorylase; AST, aspartate aminotransferase; MDH, malate dehydrogenase; MAT, methionine adenosyltransferase; and Apo, apolipoprotein.
The scan data (tiff file) were input to The Discovery Series[TM] PDQuest 2DGE Gel Analysis software for a PC computer (Ver.
High-resolution, 2DGE separation of proteins is a key step in performing a comparison between human pituitary proteomes.
Use of a commercial IPG strip (18-cm IPGstrip pH 3-10 NL) for the first-dimension IEF, a vertical multigel electrophoresis system (can analyze up to 12 gels at a time) for second-dimension SDS-PAGE, and an appropriate amount of protein loaded on the gel (~70 [micro]g) (13,14) allowed us to obtain a high-resolution, high-reproducibility 2DGE pattern of a human pituitary proteome.
The experimental pI values and molecular weights of each differential spot in Table 3 were obtained by inputting the standard 2DGE markers (Fig.
In this study, we found that most of the differential spots found across gender, age, and race were low-intensity 2DGE spots.
Comparative proteomics based on 2DGE, PDQuest analysis, and MS (15-18) provides a powerful tool to accurately analyze a human pituitary proteome across gender, age, and race.