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Proteome Science

, 10:54

First Online: 06 September 2012Received: 12 July 2012Accepted: 21 August 2012

Abstract

BackgroundGel-based proteomic is a popular and versatile method of global protein separation and quantification. However, separation of basic protein still represents technical challenges with recurrent problems of resolution and reproducibility.

ResultsThree different protocols of protein loading were compared using MCF7 cells proteins. In-gel rehydration, cup-loading and paper-bridge loading were first compared using 6–11 IPG strips, as attempted, in-gel rehydration gave large horizontal steaking; paper-bridge loading displayed an interesting spot resolution, but with a predominant loss of material; cup-loading was selected as the most relevant method, but still needing improvement. Twelve cup-loading protocols were compared with various strip rehydration, and cathodic wick solutions. Destreak appeared as better than DTT for strip rehydration; the use of isopropanol gave no improvement. The best 2DE separation was observed with cathodic wicks filled with rehydration solution complemented with DTT. Paper-bridge loading was finally analyzed using non-limited samples, such as bovine milk. In this case, new spots of basic milk proteins were observed, with or without paper wicks.

ConclusionAccording to this technical study of basic protein focalization with IPG strips, the cup-loading protocol clearly displayed the best resolution and reproducibility: strips were first rehydrated with standard solution, then proteins were cup-loaded with destreak reagent, and focalisation was performed with cathodic wicks filled with rehydration solution and DTT. Paper-bridge loading could be as well used, but preferentially with non-limited samples.

KeywordsTwo-dimensional electrophoresis Proteomic methods Isoelectric focusing IPG Cup-loading Paper bridge loading Basic proteins AbbreviationsRBRehydration buffer

TUCThiourea Urea CHAPS.

Electronic supplementary materialThe online version of this article doi:10.1186-1477-5956-10-54 contains supplementary material, which is available to authorized users.

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Autor: Jordane Dépagne - François Chevalier

Fuente: https://link.springer.com/







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