Dwuwymiarowa elektroforeza żelowa: od eksperymentu po profile ekspresji. Część pierwsza - eksperyment

Suchwałko, A.; Podbielska, H.

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Dwuwymiarowa elektroforeza żelowa: od eksperymentu po profile ekspresji. Część pierwsza - eksperyment

Autorzy

Suchwałko A. , Podbielska H.

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Warianty tytułu

Two-dimensional gel electrophoresis: from experiment to protein expresion profiles. Part one - experiment

Języki publikacji

Abstrakty

Dwuwymiarowa (dwukierunkowa) elektroforeza żelowa (2-DE) jest metodą znaną od lat 70. poprzedniego stulecia. Zainteresowanie badaczy metodą 2-DE wynika z możliwości separacji nawet kilku tysięcy białek w jednym żelu, co pozwala na ich detekcję i identyfikację. Dzięki wysokiej rozdzielczości wyników separacji możliwe staje się ustalenie roli biologicznej poszczególnych białek czy odkrywanie wpływu czynników zewnętrznych na organizmy żywe na poziomie proteomu. Metoda 2-DE wciąż ewoluuje. W ostatniej dekadzie nastąpił ogromny postęp w sposobie przeprowadzania eksperymentów z dużą powtarzalnością. Zmieniło się też całkowicie podejście do analizy obrazów żeli i wykorzystywane do tego celu algorytmy. Ze względu na tak szybki rozwój technik dwuwymiarowej elektroforezy żelowej autorzy postanowili przedstawić obecny stan wiedzy w tym zakresie. Praca składa się z dwu części. W pierwszej opisano zmiany, jakie zaszły w technikach przeprowadzania eksperymentów elektroforetycznych. Druga część skupia się na analizie obrazów uzyskanych za pomocą metody 2-DE, z uwzględnieniem zmian w schemacie analizy. Pierwsza część pracy koncentruje się na eksperymencie dwuwymiarowej elektroforezy żelowej. Opisano poszczególne kroki eksperymentu: od przygotowania próbki, przez ładowanie próbek do pierwszego wymiaru, IEF, SDS-PAGE, barwienie żeli, aż po zapis obrazów żeli. Praca nie porusza szczegółów biochemicznych eksperymentu. Opisane zostały również formaty plików, technika DIGE, wpływ eksperymentu na dalszą analizę, modyfikacje posttranslacyjne oraz pozyskanie próbek protein z żeli do ewentualne identyfikacji za pomocą innych technik proteomicznych.

Two-dimensional gel electrophoresis (2-DE) is a method commonly used since seventies of the previous century. It owns its non weakening interest of researchers because of the possibility of separation even a few thousands of proteins in one gel, what allows for protein detection and identification. Thanks to high resolution of separation results, it is possible to determine a biological role of particular proteins or to discover influence of external factors on living organisms on the proteome level. Though 2-DE is still evolving. In the last decade, a great progress has been made in the way of performing experiments and their reproducibility. Approach to the analysis of gel images and algorithms used for the analysis have been entirely changed. For the reason of such instant changes of methods and techniques of the two-dimensional gel electrophoresis, the authors decided to describe state of the art. This work consists of two parts. The first part describes changes that have been made in performing of electrophoretic experiments. The second part concentrates on the analysis of obtained images, with emphasis the analysis workflow. The first part focuses on the experiment of the dwo-dimensional gel electrophoresis. Description of particular steps of the experiment is given: from the sample preparation, through loading of samples for the first dimension, IEF, SDS-PAGE, gels staining, to recording of gel images. This work does not bring up biochemical details of the experiment. File formats, DIGE technology, influence of the expriment on the further analysis, posttraslational modifications and acquisition of protein samples from gels for identification using other proteomic techniques have been described in detail.

Słowa kluczowe

elektroforeza żelowa 2DE eksperyment analiza obrazu schemat analizy 2-DE

gel electrophoresis 2-DE experiment image analysis 2-DE analysis workflow

Wydawca

Jacek Doskocz

Czasopismo

Acta Bio-Optica et Informatica Medica. Inżynieria Biomedyczna

Rocznik

2010

Tom

Vol. 16, nr 3

Strony

285--292

Opis fizyczny

Bibliogr. 50 poz.

Twórcy

autor

Suchwałko A.

autor

Podbielska H.

Instytut Inżynierii Biomedycznej i Pomiarowej, Wydział Podstawowych Problemów Techniki, Politechnika Wrocławska, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, tel. +48 (71) 320 28 25, agnieszka.suchwalko@pwr.wroc.pl

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