Difference gel electrophoresis. A single gel method for detecting changes in protein extracts

Ünlü, Mustafa; Morgan, Mary E.; Minden, Jonathan S.

doi:10.1002/elps.1150181133

Cited by 1,944 publications

(445 citation statements)

References 14 publications

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“…Differential In-Gel Electrophoresis; DIGE [8][9][10], or saturation labeling [8;11]) due to its potential for quantitative accuracy and sensitivity. The advantages of this approach include the opportunity for multiplexing samples and inclusion of internal standards, the potential for detecting low abundance proteins, the ability to realize relative and absolute linear quantification (once the protein is identified), and the potential for real-time monitoring of electrophoresis.…”

Section: Introductionmentioning

confidence: 99%

Saturation fluorescence labeling of proteins for proteomic analyses

Pretzer

Wiktorowicz

2008

Analytical Biochemistry

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We present here an optimized and cost-effective approach to saturation fluorescence labeling of protein thiols for proteomic analysis. We investigated a number of conditions and reagent concentrations including a disulfide reducing agent (TCEP), pH, incubation time, linearity of labeling, and saturating dye: protein thiol ratio with protein standards to gauge specific and nonspecific labeling. Efficacy of labeling under these conditions was quantified using specific fluorescence estimation, defined as the ratio of fluorescence pixel intensities and Coomassie-stained pixel intensities of bands after digital imaging. Factors leading to specific vs. non-specific labeling in the presence of thiourea are also discussed.We have found that reproducible saturation of available Cys residues of the proteins used as labeling standards (human carbonic anhydrase I, enolase, α-lactalbumin) is achieved at 50-100-fold excess of the uncharged maleimide-functionalized BODIPY™ dyes over Cys. We confirm our previous findings and those of others that the maleimide dyes are not impacted by the presence of 2M thiourea. Moreover, we establish that 2 mM TCEP used as reductant is optimal. We also establish further that labeling is optimal at pH 7.5 and complete after 30 min. Low non-specific labeling was gauged by the inclusion of non-Cys containing proteins (horse myoglobin, bovine carbonic anhydrase) to the labeling mixture. We also show that the dye exhibits little to no effect on the two dimensional mobilities of labeled proteins derived from cells.

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Section: Introductionmentioning

confidence: 99%

Saturation fluorescence labeling of proteins for proteomic analyses

Pretzer

Wiktorowicz

2008

Analytical Biochemistry

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“…The DIGE makes use of fluorescent cyanine dyes, such as Cy3 and Cy5 [74][75][76][77] that differ in their wavelengths. They are used to label differentially two or more cell situations prior to 2-DE.…”

Section: Protein Quantificationmentioning

confidence: 99%

Two‐dimensional gel electrophoresis as tool for proteomics studies in combination with protein identification by mass spectrometry

2006

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The proteome analysis by 2-DE is one of the most potent methods of analyzing the complete proteome of cells, cell lines, organs and tissues in proteomics studies. It allows a fast overview of changes in cell processes by analysis of the entire protein extracts in any biological and medical research projects. New instrumentation and advanced technologies provide proteomics studies in a wide variety of biological and biomedical questions. Proteomics work is being applied to study antibiotics-resistant strains and human tissues of various brain, lung, and heart diseases. It cumulated in the identification of antigens for the design of new vaccines. These advances in proteomics have been possible through the development of advanced high-resolution 2-DE systems allowing resolution of up to 10 000 protein spots of entire cell lysates in combination with protein identification by new highly sensitive mass spectrometric techniques. The present technological achievements are suited for a high throughput screening of different cell situations. Proteomics may be used to investigate the health effects of radiation and electromagnetic field to clarify possible dangerous alterations in human beings.

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“…One of the most notable advancements in proteome analysis made over recent years has been the development of an approach known as 2-D differential gel electrophoresis (2D-DIGE) (Unlü et al, 1997). This technique is an invaluable approach for analyzing global changes in protein expression patterns within a certain system (e.g.…”

Section: Advancements and Current Status Of 2-dementioning

confidence: 99%

“…by covalently labeling two or three different protein samples with different fluorescent dyes that have distinct excitation and emission characteristics, they may be mixed and analyzed together on the same 2-D gel ( Fig. 1) (Unlü et al, 1997;Marouga et al, 2005). Gels are visualized using a commercial fluorescent gel scanner/densitometer to quantify the fluorescence intensity, which is proportional to the amount of (labeled) protein present in each spot.…”

Section: Advancements and Current Status Of 2-dementioning

confidence: 99%

Two-dimensional gel electrophoresis in bacterial proteomics

et al. 2012

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Two-dimensional gel electrophoresis (2-DE) is a gel-based technique widely used for analyzing the protein composition of biological samples. It is capable of resolving complex mixtures containing more than a thousand protein components into individual protein spots through the coupling of two orthogonal biophysical separation techniques: isoelectric focusing (first dimension) and polyacrylamide gel electrophoresis (second dimension). 2-DE is ideally suited for analyzing the entire expressed protein complement of a bacterial cell: its proteome. Its relative simplicity and good reproducibility have led to 2-DE being widely used for exploring proteomics within a wide range of environmental and medically-relevant bacteria. Here we give a broad overview of the basic principles and historical development of gel-based proteomics, and how this powerful approach can be applied for studying bacterial biology and physiology. We highlight specific 2-DE applications that can be used to analyze when, where and how much proteins are expressed. The links between proteomics, genomics and mass spectrometry are discussed. We explore how proteomics involving tandem mass spectrometry can be used to analyze (post-translational) protein modifications or to identify proteins of unknown origin by de novo peptide sequencing. The use of proteome fractionation techniques and non-gel-based proteomic approaches are also discussed. We highlight how the analysis of proteins secreted by bacterial cells (secretomes or exoproteomes) can be used to study infection processes or the immune response. This review is aimed at non-specialists who wish to gain a concise, comprehensive and contemporary overview of the nature and applications of bacterial proteomics.

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Difference gel electrophoresis. A single gel method for detecting changes in protein extracts

Cited by 1,944 publications

References 14 publications

Saturation fluorescence labeling of proteins for proteomic analyses

Saturation fluorescence labeling of proteins for proteomic analyses

Two‐dimensional gel electrophoresis as tool for proteomics studies in combination with protein identification by mass spectrometry

Two-dimensional gel electrophoresis in bacterial proteomics

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