Protein stains for proteomic applications:<b><i>Which, when, why?</i></b>

Miller, Ingrid; Crawford, Janet; Gianazza, Elisabetta

doi:10.1002/pmic.200600323

Cited by 214 publications

(157 citation statements)

References 201 publications

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“…Colorimetric methods include CBB and variants, such as Colloidal (C)-CBB or blue silver (C-CBB modified), silver-staining (there are more than 100 variants) and zinc or imidazole-zinc staining. Most of them have linear responses over a very limited range (maximum two orders of magnitude) due to saturation effects that make them unable to cover the great variation in protein concentration of a sample (see discussion reviewed by [97][98][99][100]). CBB-based stainings are simple to use and compatible with MS with a detection limit of around 8-100 ng/spot or 1-100 ng/spot for blue silver [101].…”

Section: Protein Visualizationmentioning

confidence: 99%

“…Postelectrophoretic protein visualization is most frequently obtained by colorimetric and fluorescent staining methods. The critical factors of staining methods are their sensitivity, reproducibility and the linear range of detection (for a review see [97,98]). Colorimetric methods include CBB and variants, such as Colloidal (C)-CBB or blue silver (C-CBB modified), silver-staining (there are more than 100 variants) and zinc or imidazole-zinc staining.…”

Section: Protein Visualizationmentioning

confidence: 99%

“…Fluorescence staining methods are being increasingly used due to their comparatively wide linear dynamic range (. 10 3 ), good sensitivity (1-2 ng protein/spot) and ease of use (for recent reviews [97,98]). The overall quality and relative cost of five commercially available fluorescent stains, Krypton, Deep Purple, Rubeo, Flamingo and the most popular used stain, Sypro Ruby dye were recently evaluated by Harris et al 2007 [97].…”

Section: Protein Visualizationmentioning

confidence: 99%

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Two‐dimensional gel electrophoresis and mass spectrometry for biomarker discovery

Penque

2009

Proteomics Clinical Apps

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The proteome project, initiated in 1995, was made possible by 2-DE combined with MS. The project main objective was and remains the identification of all proteins expressed by a cell, tissue or organism in a given time and condition. Following this objective, the global profiling of proteins in health versus pathological state by the 2-DE/MS-based proteomic approach has contributed to the elucidation of the basic mechanisms of disease by discovering candidate disease biomarkers and disease targets for new drug development. This review will briefly summarize the historical evolution of 2-DE up to today, and review 2-DE/MS technology and its specific methods of study of immunoresponse (immunoproteomics), PTM of proteins, complex proteinprotein interactions (interactome), the proteome of cell membrane and intracellular proteome turnover in disease biomarker discovery. 2-DE historical evolutionGlobal profiling of proteins in healthy versus pathological states is a strategy to discover biomarkers for early diagnostics, disease progression, treatment response and novel targets for therapeutic drugs development. The idea of making an inventory of all the proteins in a cell, tissue or organism with normal or abnormal physiology, was (re)initiated in the middle of the 1990s with the proteome project [1]. Since then, many technologies to make the analysis of the proteome a reality have been united.The perfect technological 'marriage' that made the proteome project feasible was between high resolution 2-DE for separation of large numbers of proteins and MS for identification and characterization of the proteins displayed on this gel [2]. However, before this happy union occured, science and technology devoted to protein study had a long journey until 2-DE and MS combination was possible (Fig. 1). It started in the last century, in 1937, when Tiselius [3] introduced electrophoresis technique as a modification of Reiner's early concept (1927) [4] to analyse a complex mixture of proteins such as serum proteins. Only 20 years later, in 1959, electrophoresis on a gel support formed by crosslinked polymerization of Cyanogum 41, a commercial name for two organic monomers, acrylamide and the crosslinking agent, N,N1-methylenebisacrylamide, was shown to be useful and was named PAGE by Raymond and Weintraub [5]. The adaptation of polyacrylamide gel to IEF in a stable pH gradient, developed by Svensson in 1962 [6], was possible by the end of the 1960s [7][8][9]. About this time, the 2-D by combining IEF to separate undenatured proteins according to their charge and gradient SDS-PAGE for a second separation, according to their molecular mass, was for the first time introduced by Kenrick and Margolis (1970) [10]. Only in 1975, was IEF in denaturing conditions, as known today, used to follow the 2-D PAGE and it was described in three independent works [11][12][13]. The most powerful demonstraCorrespondence: Dr. Deborah Penque, Laboratório de Proteó-mica, Departamento de Genética, Instituto Nacional de Saúde, Dr. Ricardo Jorge, I.P., A...

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Section: Protein Visualizationmentioning

confidence: 99%

Section: Protein Visualizationmentioning

confidence: 99%

Section: Protein Visualizationmentioning

confidence: 99%

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Two‐dimensional gel electrophoresis and mass spectrometry for biomarker discovery

Penque

2009

Proteomics Clinical Apps

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“…By using these two new methodologies, genomics and proteomic analysis, together in combination with cell engineering strategies we are one step closer towards a better understanding of cellular behavior during bioprocessing. Two dimensional polyacrylamide gel electrophoresis (2D-PAGE or 2DE) is the most common proteomic technique and with the recent advent of 2D-differential gel electrophoresis (DIGE) (Lilley and Friedman 2004) combined with advances in quantitative analysis, in image warping and spot detection algorithms (Miller et al 2006), followed by MALDI-TOF (matrix-assisted laser-desorption/ionization time-of-flight) mass spectrometry (MS) or LC-MS/MS (liquid chromatography) for protein identification (Aebersold and Mann 2003; Gorg et al 2004), a powerful proteomic tool has been generated. In genomics the use of high-density oligonucleotide GeneChip microarrays for transcriptomics analysis has become a common method to analyse gene expression.…”

Section: Genomics and Proteomics-tools For Indirect Cell Engineeringmentioning

confidence: 99%

Using cell engineering and omic tools for the improvement of cell culture processes

et al. 2007

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Significant strides have been made in mammalian cell based biopharmaceutical process and cell line development over the past years. With several established mammalian host cell lines and expression systems, optimization of selection systems to reduce development times and improvement of glycosylation patterns are only some of the advances being made to improve cell culture processes. In this article, the advances pertaining to cell line development and cell engineering strategies are discussed. An overview of the cell engineering strategies to enhance cellular characteristics by genetic manipulation are illustrated, focusing on the use of genomics and proteomics tools and their application in such endeavors. Included in this review are some of the early studies using the 'omic' technique to understand cellular mechanisms of product synthesis and secretion, apoptosis, cell proliferation and the influence of the physicochemical environment. The article highlights the significance of integrating genomics and proteomics data with the vast amounts of bioprocess data for improved analysis of the biological pathways involved. Further improvements of the techniques and methodologies used are needed but ultimately, the new cell engineering strategies should provide great insight into the regulatory networks within the cell in a bioprocess environment and how to manipulate them to increase overall productivity.

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“…For example, the UV absorbance of aromatic amino acids at 280 nm, colorimetry methods such as the Bradford and Lowry assays, the bicinchoninic acid method, and gel staining with Coomassie Brilliant Blue R-250 or silver are well established and used for the quantification of crude proteins. [2][3][4][5][6][7] These are simple methods that are appropriate for many samples; however, they can have problems, such as fluctuations of the absorption depending on the protein and the presence of interfering substances.…”

Section: Introductionmentioning

confidence: 99%

Quantification of Proteins by Measuring the Sulfur Content of Their Constituent Peptides by Means of Nano HPLC-ICPMS

2014

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8 ICPL (isotope-coded protein label), 9 and iTRAQ (isobaric tag for relative and absolute quantification). 10 These methods require not only a specific antibody, but also an amino acid-specific labeling reagents. New protein quantification methods that do 2014 © The Japan Society for Analytical Chemistry † To whom correspondence should be addressed. E-mail: nfuruta@chem.chuo-u.ac.jp Y. S. present address: Faculty of Life and Environmental Science, Department of Regional Environmental Science, Shimane University, 1060 Nishikawatsu-cho, Shimane, Japan. Quantification of Proteins by Measuring the Sulfur Content of Their Constituent Peptides by Means of Nano HPLC-ICPMSYoshinari SUZUKI, Ayumi NOBUSAWA, and Naoki FURUTA † Faculty of Science and Engineering, Department of Applied Chemistry, Chuo University, Bunkyo, Japan The sulfur (S) concentrations of three peptides were determined by using nano HPLC-ICPMS under a flow of O2 in an octapole reaction cell, and the determined values showed a good agreement with theoretical values. This method was then applied to trypsin-digested peptides from human albumin for protein quantification. Assigning of the number of S atoms in each peptide/peak and the tryptic digestion efficiency were important for protein quantification. The number of S atoms in each peptide/peak was assigned by using verification scores that gave the lowest standard deviation of the peptide S concentration and the highest S recovery. The peptide concentrations were calculated as the ratio of the S concentration/the number of S atoms in the peptide/peak. The tryptic digestion efficiency was calculated as the sum of the S concentration in the mono-peptides divided by the total S concentration in a native polyacrylamide gel electrophoresis (PAGE) band before tryptic digestion. Our result indicates that a protein can be quantified through peptide quantification, after taking into account the tryptic digestion efficiency, via S quantification using ICPMS.

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Protein stains for proteomic applications:Which, when, why?

Cited by 214 publications

References 201 publications

Two‐dimensional gel electrophoresis and mass spectrometry for biomarker discovery

Two‐dimensional gel electrophoresis and mass spectrometry for biomarker discovery

Using cell engineering and omic tools for the improvement of cell culture processes

Quantification of Proteins by Measuring the Sulfur Content of Their Constituent Peptides by Means of Nano HPLC-ICPMS

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