Technical updates to basic proteins focalization using IPG strips

Proteome Science

François Chevalier¹  Jordane Dépagne¹ 

[1] Proteomic Laboratory, iRCM / DSV / CEA, Fontenay aux Roses, France

关键词: Basic proteins;    Paper bridge loading;    Cup-loading;    IPG;    Isoelectric focusing;    Proteomic methods;    Two-dimensional electrophoresis;   

Others  :  817191
DOI  :  10.1186/1477-5956-10-54

 received in 2012-07-12, accepted in 2012-08-21,  发布年份 2012

【 摘 要 】

Background

Gel-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.

Results

Three 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.

Conclusion

According 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.

【 授权许可】

   
2012 Dépagne and Chevalier; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Van den Bergh G, Arckens L: High Resolution Protein Display by Two-Dimensional Electrophoresis. Curr Anal Chem 2009, 5:106-115.
• [2]Chevalier F: Highlights on the capacities of “Gel-based” proteomics. Proteome Science 2010, 8:23. BioMed Central Full Text
• [3]Rabilloud T, Lelong C: Two-dimensional gel electrophoresis in proteomics: A tutorial. J Proteomics 2011, 74:1829-1841.
• [4]Luche S, Diemer H, Tastet C, Chevallet M, Van Dorsselaer A, Leize-Wagner E, Rabilloud T: About thiol derivatization and resolution of basic proteins in two-dimensional electrophoresis. Proteomics 2004, 4:551-561.
• [5]Görg A, Drews O, Lück C, Weiland F, Weiss W: 2-DE with IPGs. Electrophoresis 2009, 30:S122-S132.
• [6]Hoving S, Gerrits B, Voshol H, Müller D, Roberts RC, van Oostrum J: Preparative two-dimensional gel electrophoresis at alkaline pH using narrow range immobilized pH gradients. Proteomics 2002, 2:127-134.
• [7]Olsson I, Larsson K, Palmgren R, Bjellqvist B: Organic disulfides as a means to generate streak-free two-dimensional maps with narrow range basic immobilized pH gradient strips as first dimension. Proteomics 2002, 2:1630-1632.
• [8]Kask L, Larsson K, Bjellqvist B: Elimination of basic gaps at high pH values in 2‐DE. Proteomics 2009, 9:5558-5561.
• [9]Kane LA, Yung CK, Agnetti G, Neverova I, Van Eyk JE: Optimization of paper bridge loading for 2-DE analysis in the basic pH region: application to the mitochondrial subproteome. Proteomics 2006, 6:5683-5687.
• [10]Chevalier F: Analytical Methods Electrophoresis. In Encyclopedia of Dairy Sciences. San Diego: Academic; 2011:185-192.
• [11]Fox PF, McSweeney PL: Milk proteins. In Dairy Chemistry and Biochemistry. London: BA; 1998:150-169.
• [12]Poth AG, Deeth HC, Alewood PF, Holland JW: Analysis of the Human Casein Phosphoproteome by 2-D Electrophoresis and MALDI-TOF/TOF MS Reveals New Phosphoforms. J Proteome Res 2008, 7:5017-5027.
• [13]Holland JW, Deeth HC, Alewood PF: Analysis of disulphide linkages in bovine kappa-casein oligomers using two-dimensional electrophoresis. Electrophoresis 2008, 29:2402-2410.
• [14]Chevalier F, Chobert JM, Dalgalarrondo M, Haertle T: Characterization of the Maillard reaction products of beta-lactoglobulin glucosylated in mild conditions. J Food Biochem 2001, 25:33-55.
• [15]Chevalier F, Hirtz C, Sommerer N, Kelly AL: Use of Reducing/Nonreducing Two-Dimensional Electrophoresis for the Study of Disulfide-Mediated Interactions between Proteins in Raw and Heated Bovine Milk. J Agric Food Chem 2009, 57:5948-5955.
• [16]Chevalier F, Kelly AL: Proteomic Quantification of Disulfide-Linked Polymers in Raw and Heated Bovine Milk. J Agric Food Chem 2010, 58:7437-7444.
• [17]Chevalier F: Milk Proteins Proteomics. In Encyclopedia of Dairy Sciences. San Diego: Academic; 2011:843-847.
• [18]Chevalier F, Depagne J, Hem S, Chevillard S, Bensimon J, Bertrand P, Lebeau J: Accumulation of Cyclophilin A isoforms in conditioned medium of irradiated breast cancer cells. Proteomics 2012, 12:1756-1766.
• [19]Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976, 72:248-254.
• [20]Chevalier F, Rofidal V, Vanova P, Bergoin A, Rossignol M: Proteomic capacity of recent fluorescent dyes for protein staining. Phytochemistry 2004, 65:1499-1506.
• [21]Chevalier F, Centeno D, Rofidal V, Tauzin M, Martin O, Sommerer N, Rossignol M: Different impact of staining procedures using visible stains and fluorescent dyes for large-scale investigation of proteomes by MALDI-TOF mass spectrometry. J Proteome Res 2006, 5:512-520.