Two-dimensional fluorescence difference gel electrophoresis for comparative proteomics profiling

Tannu, Nilesh S; Hemby, Scott E.

doi:10.1038/nprot.2006.256

Cited by 166 publications

(128 citation statements)

References 66 publications

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“…23,36,37 Aliquots of cytosolic proteins were diluted in 400 μl of rehydration buffer and increased to a final volume of 450 μl with DeStreak rehydration buffer (GE Healthcare, Piscataway, NJ, USA). Isoelectric focusing was performed using Immobiline DryStrips (240×3×0.5 mm, pH 4-7 linear; GE Healthcare) on an Ettan IPGphor apparatus (GE Healthcare).…”

Section: Protein Isolation and Fractionationmentioning

confidence: 99%

“…Isoelectric focusing was performed using Immobiline DryStrips (240×3×0.5 mm, pH 4-7 linear; GE Healthcare) on an Ettan IPGphor apparatus (GE Healthcare). 23,36,37 Gel image analysis-Image analysis was conducted as described previously, 23,36,37 using a Typhoon 9400 scanner (GE Healthcare) to scan all gels at 100 μm resolution. The photomultiplier tube was set to ensure maximum pixel intensity of 85 000-95 000 for all the images in every gel.…”

Section: Protein Isolation and Fractionationmentioning

confidence: 99%

“…Experiment 1: Differential native protein expression using 2D-DIGE Cyanine dye labeling-Minimal labeling of the lysine residues was achieved by reaction with cyanine dyes, as described previously. 23,36,37 Briefly, a normalization (pooled) sample was prepared by combining 50 μg from each sample. The labeling of 50 μg of protein sample (each sample) was optimized by labeling with 200 pmol of appropriate dye (suspended in > 99.5% pure dimethylformamide).…”

Section: Protein Isolation and Fractionationmentioning

confidence: 99%

“…The protein spot matches performed in the automatic mode were also confirmed manually for all the gels. 37 To visualize the proteome from this specific pH and mass range, gels were stained with Sypro Ruby stain overnight. The excess stain was removed by 10% methanol and 6% glacial acetic acid for 20 min.…”

Section: Experiments 2: Phosphoproteome Gel Staining and Image Analysismentioning

confidence: 99%

“…The average ratio and also the corresponding student's t-test value for each protein spot was calculated based on all gel images in the DeCyder BVA mode. 37 Selected spots were isolated using the Ettan Spot Handling Workstation (GE Healthcare) and picked proteins were prepared for MS analysis as described in the section below entitled in-gel trypsin digestion.…”

Section: Protein Isolation and Fractionationmentioning

confidence: 99%

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Integrative proteomic analysis of the nucleus accumbens in rhesus monkeys following cocaine self-administration

2008

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The reinforcing effects and long-term consequences of cocaine self-administration have been associated with brain regions of the mesolimbic dopamine pathway, namely the nucleus accumbens (NAc). Studies of cocaine-induced biochemical adaptations in rodent models have advanced our knowledge; however, unbiased detailed assessments of intracellular alterations in the primate brain are scarce, yet essential, to develop a comprehensive understanding of cocaine addiction. To this end, two-dimensional difference in gel electrophoresis (2D-DIGE) was used to compare changes in cytosolic protein abundance in the NAc between rhesus monkeys selfadministering cocaine and controls. Following image normalization, spots with significantly differential image intensities (P < 0.05) were identified, excised, trypsin digested and analyzed by matrix-assisted laser-desorption ionization time-of-flight time-of-flight (MALDI-TOF-TOF). In total, 1098 spots were subjected to statistical analysis with 22 spots found to be differentially abundant of which 18 proteins were positively identified by mass spectrometry. In addition, approximately 1000 protein spots were constitutively expressed of which 21 proteins were positively identified by mass spectrometry. Increased levels of proteins in the cocaine-exposed monkeys include glial fibrillary acidic protein, syntaxin-binding protein 3, protein kinase C isoform, adenylate kinase isoenzyme 5 and mitochondrial-related proteins, whereas decreased levels of proteins included β-soluble N-ethylmaleimide-sensitive factor attachment protein and neural and non-neural enolase. Using a complimentary proteomics approach, the differential expression of phosphorylated proteins in the cytosolic fraction of these subjects was examined. Two-dimensional gel electrophoresis (2DGE) was followed by gel staining with Pro-Q Diamond phosphoprotein gel stain, enabling differentiation of approximately 150 phosphoprotein spots between the groups. Following excision and trypsin digestions, MALDI-TOF-TOF was used to confirm the identity of 15 cocaine-altered phosphoproteins. Significant increased levels were detected for γ-aminobutyric acid type A receptor-associated protein 1, 14-3-3 γ-protein, glutathione S-transferase and brain-type aldolase, whereas significant decreases were observed for β-actin, Rab GDP-dissociation inhibitor, guanine deaminase, peroxiredoxin 2 isoform b and Results from these studies indicate coordinated dysregulation of proteins related to cell structure, signaling, metabolism and mitochondrial function. These data extend and compliment previous studies of cocaine-induced biochemical alterations in human post-mortem brain tissue, using an animal model that closely recapitulates the human condition and provide new insight into the molecular basis of the disease and potential targets for pharmacotherapeutic intervention.

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Section: Protein Isolation and Fractionationmentioning

confidence: 99%

Section: Protein Isolation and Fractionationmentioning

confidence: 99%

Section: Protein Isolation and Fractionationmentioning

confidence: 99%

Section: Experiments 2: Phosphoproteome Gel Staining and Image Analysismentioning

confidence: 99%

Section: Protein Isolation and Fractionationmentioning

confidence: 99%

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Integrative proteomic analysis of the nucleus accumbens in rhesus monkeys following cocaine self-administration

2008

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Common Proteomic Changes in the Hippocampus in Schizophrenia and Bipolar Disorder and Particular Evidence for Involvement of Cornu Ammonis Regions 2 and 3

Föcking

Dicker²,

English³

et al. 2011

Arch Gen Psychiatry

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Difference Gel Electrophoresis ( DIGE )

Granlund

Hall

Schröder

2009

Encyclopedia of Life Sciences

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One of the largest challenges in proteomics today is to be able to quantify the composition and amount of proteins found in a specific cell or tissue at a defined time point. Difference gel electrophoresis (DIGE) is a gel electrophoresis‐based technique for protein quantification in complex mixtures. In DIGE the high resolution of two‐dimensional gel electrophoresis is combined with the excellent dynamic range obtained by fluorescent tag labelling of protein samples. The output of DIGE experiments provides information about how many proteins display changed expression levels on a specific treatment. In addition, proteins of interest can be excised and identified with conventional mass spectrometry techniques and further analysed by other biochemical methods. Key concepts: DIGE is a gel‐based technique for relative protein quantification in complex protein samples. Experimental design, sample preparation and data analysis are all critical steps of a DIGE experiment. Labelling of protein samples is performed by either a minimal or saturation labelling procedure. Use of an internal standard minimizes gel to gel variation and provides increased power to the experiment. For protein identification poststaining of gels is necessary when a minimal labelling procedure has been used. DIGE can also be used for other applications such as native gel electrophoresis of protein complexes. The DIGE technique is constantly being improved and continues to be an important method in functional proteomics.

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Two-dimensional fluorescence difference gel electrophoresis for comparative proteomics profiling

Cited by 166 publications

References 66 publications

Integrative proteomic analysis of the nucleus accumbens in rhesus monkeys following cocaine self-administration

Integrative proteomic analysis of the nucleus accumbens in rhesus monkeys following cocaine self-administration

Common Proteomic Changes in the Hippocampus in Schizophrenia and Bipolar Disorder and Particular Evidence for Involvement of Cornu Ammonis Regions 2 and 3

Difference Gel Electrophoresis ( DIGE )

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