Detergents and Chaotropes for Protein Solubilization before Two-Dimensional Electrophoresis

Because of the outstanding separating capabilities of two-dimensional electrophoresis for complete proteins, it would be advantageous to be able to apply it to all types of proteins. Unfortunately, severe solubility problems hamper the analysis of many classes of proteins, but especially membrane proteins. These problems arise mainly in the extraction and isoelectric focusing steps, and solutions are sought to improve protein solubility under the conditions prevailing during isoelectric focusing. These solutions deal mainly with chaotropes and new detergents, which are both able to enhance protein solubility. The input of these compounds in proteomics analysis of membrane proteins is discussed, as well as future directions.Comment: link to publisher's site http://biomed.humanapress.com

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

L'INDAGINE SUI PALAZZI PRETORI SOTTO IL GOVERNO DI PIETRO LEOPOLDO

La tesi si basa sull’analisi di tre filze conservate nell’Archivio di Stato di Firenze ascrivibili agli anni ’70 e ’80 del XVIII secolo nelle quali si descrive la “riduzione di buon grado di palazzi pretori di varie Cancellerie”. I tre faldoni contengono una serie di relazioni tecniche, talvolta corredate da pregevole documentazione grafica, e un abbondante epistolario che ha per oggetto la visita e sistemazione dei palazzi pretori dell’area fiorentina, secondo quanto prescritto dalla legge di riforma dei governi provinciali del 1772, nella quale si richiede “che tutti i palazzi pretori siano posti in buon grado”. L’idea di intraprendere una campagna di indagine sistematica dei palazzi, nasce tuttavia da una successiva proposta del soprassindaco della Camera delle comunità di Firenze, Giovanni Battista Nelli, avallata dal sovrano con successivo rescritto del 14 agosto 1777, ragione per la quale essa riguarda la sola giurisdizione fiorentina, con l’unica eccezione di Portoferraio. Il presente lavoro si sofferma in particolare sull’analisi di sette casi, Certaldo, Castelfiorentino, Fucecchio, Marradi, Montepulciano, Monte San Savino e Pontremoli, selezionati in base alla quantità di informazioni e al materiale grafico presente. Per ciascuno viene fatta una breve descrizione dell’aspetto odierno dell’edificio e vengono forniti alcuni cenni storici; successivamente si espone la vicenda relativa restauro settecentesco, ricavata dalla lettura delle fonti, ponendo particolare attenzione all’illustrazione dei progetti elaborati

La narración como encuentro con el Otro en El Hablador de Mario Vargas LLosa

El presente texto se propone mostrar la manera en la que Vargas Llosa trata el tema histórico del encuentro-desencuentro entre la cultura occidental y la cultura indígena en América Latina. La novela cuenta el clásico intento de un personaje de convertirse en el Otro y muestra cómo la única manera para lograrlo sea la de escuchar las historias y la cosmogonia del Otro y volver a contar, re-elaborándolas, las historia escuchadas y al mismo tiempo traducir a su lenguaje narrativo la propia tradición.The paper aims to explain the perspective from which Vargas Llosa in The Storyteller faces the historical theme of the clash-encounter between Western culture and Indian culture in Latin America. The novel tells the classic attempt of a character to become Another and shows how the only way to achieve it is to hear the stories and the cosmogony of the Other, to re-tell the story heard and at the same time to translate into the Other narrative language one's own tradition

Mitochondrial proteomics: analysis of a whole mitochondrial extract with two-dimensional electrophoresis

Mitochondria are complex organelles, and their proteomics analysis requires a combination of techniques. The emphasis in this chapter is made first on mitochondria preparation from cultured mammalian cells, then on the separation of the mitochondrial proteins with two-dimensional electrophoresis (2DE), showing some adjustment over the classical techniques to improve resolution of the mitochondrial proteins. This covers both the protein solubilization, the electrophoretic part per se, and the protein detection on the gels, which makes the interface with the protein identification part relying on mass spectrometry

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

Organelle proteomics.

International audienceThis unit describes strategies for studying the proteomes of organelles, which is one example of targeted proteomics. It relies heavily on previously published units dealing with organelle preparation, protein solubilization, and proteomics techniques. A specific commentary for organelle proteomics is provided. Specific protocols for the isolation of nuclei from various sources (cell cultures, tissues) are also provided

Les bibliothèques et la deuxième naissance Loin de porter du cinéma

Loin de porter concurrence aux exploitants de cinéma en salle, l’offre développée par les médiathèques a souvent pallié les déficiences du réseau commercial. La preuve en Nord – Pas-de-Calais où, dans l’urgence, des formations ont même émergé de façon informelle

Escapades bibliothéconomiques en Flandres et Pas-de-Calais

En deux « escapades » automnales, le groupe régional proposait à plus d’une vingtaine de participants de visiter cinq équipements récents, un SCD et quatre médiathèques de petites villes du Nord