Technical updates to basic proteins focalization using IPG strips

Jordane Dépagne,François Chevalier

doi:10.1186/1477-5956-10-54

Jordane Dépagne, François Chevalier

Open Access

https://doi.org/10.1186/1477-5956-10-54

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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.

Highlights

Gel-based proteomic is a popular and versatile method of global protein separation and quantification
IEF in the alkaline region is considered as a challenge to separate basic proteins by 2-DE and most of gel-based proteomic studies were performed in the acidic range
In case of protein loading during strip rehydration (Figure 1A), proteins were mixed with rehydration buffer (RB) (7 M urea, 2 M thiourea, 4% CHAPS, 0.05% triton X100, 5% glycerol) complemented with 1.2% destreak reagent and 0.5% IPG buffer 6–11

Summary

Introduction

Gel-based proteomic is a popular and versatile method of global protein separation and quantification. Separation of basic protein still represents technical challenges with recurrent problems of resolution and reproducibility. Two-dimensional electrophoresis is until now one of the most widely used technique for performing functional proteomics [1]. It has the capacity to separate, visualize and quantify several thousands of proteins in a single gel from a complex biological sample, allowing the large-scale analysis of protein expression differences. IEF in the alkaline region is considered as a challenge to separate basic proteins by 2-DE and most of gel-based proteomic studies were performed in the acidic range. Basic proteins are difficult to separate in the first dimension for several reasons.

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