Silver Staining of Proteins in 2DE Gels

Silver staining detects proteins after electrophoretic separation on polyacrylamide gels. Its main positive features are its excellent sensitivity (in the low nanogram range) and the use of very simple and cheap equipment and chemicals. The sequential phases of silver staining are protein fixation, then sensitization, then silver impregnation, and finally image development. Several variants of silver staining are described here, which can be completed in a time range from 2 h to 1 day after the end of the electrophoretic separation. Once completed, the stain is stable for several weeks

Improved proteomic analysis of nuclear proteins, as exemplified by the comparison of two myelo\"id cell lines nuclear proteomes

One of the challenges of the proteomic analysis by 2D-gel is to visualize the low abundance proteins, particularly those localized in organelles. An additional problem with nuclear proteins lies in their strong interaction with nuclear acids. Several experimental procedures have been tested to increase, in the nuclear extract, the ratio of nuclear proteins compared to contaminant proteins, and also to obtain reproducible conditions compatible with 2D-gel electrophoresis. The NaCl procedure has been chosen. To test the interest of this procedure, the nuclear protein expression profiles of macrophages and dendritic cells have been compared with a proteomic approach by 2D-gel electrophoresis. Delta 2D software and mass spectrometry analyses have allowed pointing out some proteins of interest. We have chosen some of them, involved in transcriptional regulation and/or chromatin structure for further validations. The immunoblotting experiments have shown that most of observed changes are due to post-translational modifications, thereby a exemplifying the interest of the 2D gel approach. Finally, this approach allowed us to reach not only high abundance nuclear proteins but also lower abundance proteins, such as the HP1 proteins and reinforces the interest of using 2DE-gel in proteomics because of its ability to visualize intact proteins with their modifications

Two-dimensional gel electrophoresis in proteomics: A tutorial

Two-dimensional electrophoresis of proteins has preceded, and accompanied, the birth of proteomics. Although it is no longer the only experimental scheme used in modern proteomics, it still has distinct features and advantages. The purpose of this tutorial paper is to guide the reader through the history of the field, then through the main steps of the process, from sample preparation to in-gel detection of proteins, commenting the constraints and caveats of the technique. Then the limitations and positive features of two-dimensional electrophoresis are discussed (e.g. its unique ability to separate complete proteins and its easy interfacing with immunoblotting techniques), so that the optimal type of applications of this technique in current and future proteomics can be perceived. This is illustrated by a detailed example taken from the literature and commented in detail. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP 2)

Glioma cell dispersion is driven by [alfa]5 integrin-mediated cell-matrix and cell-cell interactions

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Two-dimensional gel electrophoresis in proteomics: past, present and future

Two-dimensional gel electrophoresis has been instrumental in the birth and developments of proteomics, although it is no longer the exclusive separation tool used in the field of proteomics. In this review, a historical perspective is made, starting from the days where two-dimensional gels were used and the word proteomics did not even exist. The events that have led to the birth of proteomics are also recalled, ending with a description of the now well-known limitations of two-dimensional gels in proteomics. However, the often-underestimated advantages of two-dimensional gels are also underlined, leading to a description of how and when to use two-dimensional gels for the best in a proteomics approach. Taking support of these advantages (robustness, resolution, and ability to separate entire, intact proteins), possible future applications of this technique in proteomics are also mentioned

Drying colloidal systems: laboratory models for a wide range of applications

The drying of complex fluids provides a powerful insight into phenomena that take place on time and length scales not normally accessible. An important feature of complex fluids, colloidal dispersions and polymer solutions is their high sensitivity to weak external actions. Thus, the drying of complex fluids involves a large number of physical and chemical processes. The scope of this review is the capacity to tune such systems to reproduce and explore specific properties in a physics laboratory. A wide variety of systems are presented, ranging from functional coatings, food science, cosmetology, medical diagnostics and forensics to geophysics and art

The whereabouts of 2D gels in quantitative proteomics

International audienceTwo-dimensional gel electrophoresis has been instrumental in the development of proteomics. Although it is no longer the exclusive scheme used for proteomics, its unique features make it a still highly valuable tool, especially when multiple quantitative comparisons of samples must be made, and even for large samples series. However, quantitative proteomics using two-dimensional gels is critically dependent on the performances of the protein detection methods used after the electrophoretic separations. This chapter therefore examines critically the various detection methods, (radioactivity, dyes, fluorescence, and silver) as well as the data analysis issues that must be taken into account when quantitative comparative analysis of two-dimensional gels is performed

Régulation de l'activité du facteur Sigma S par la protéine Crl chez Escherichia coli

Chez Escherichia coli, la sous unité sigma RpoS contrôle l'expression des gènes de la phase stationnaire et de la réponse généralisée aux stress. La synthèse de RpoS esr régulée au niveau de la transcription, de la traduction et de la stabilité de la protéine. Malgré les travaux intensifs des dix dernières années dont le facteur sigma RpoS a fait l'objet, la régulation de l'activité de cette protéine reste encore mal connue. La protéine Crl, impliquée dans le contrôle de l'expression des curlis, permet de moduler positivement l'activité du facteur Rpos. Il a été démontré récemment, par des expériences de génétique, que crl est indispensable à une activité transcriptionnelle optimale du promoteur des curlis, mais également de certains gènes appartenant au régulon de RpoS. Il a donc été proposé que Crl agisse soit en amont, soit de concert avec RpoS. Nous montrons ici que Crl interagit directement avec RpoS in vitro. En utilisant la région promotrice des gènes codant les curlis comme modèle d'étude, nous avons mis en évidence un effet positif de Crl sur le recrutement de l'holoenzyme-RpoS au niveau du promoteur. Nous avons également montré que l'expression de Crl est augmentée lors de la transition entre la phase de croissance et la phase stationnaire. Crl s'accumule fortement dans les cellules en phase stationnaire à 30C et beaucoup moins à 37C. Crl permettrai donc un contrôle de l'activité de RpoS en fonction de la tempréature. Nos résultats suggèrent un rôle de Crl au niveau de l'association de la sous unité sigma Rpos avec l'ARN polymérase et/ou au niveau de la sélectivité entre l'holoenzyme-RpoS et l'holoenzyme-RpoD pour le promoteur cible.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF