Volume 5, Issue 9 p. 2331-2339
Regular Article

Acrylamide-agarose copolymers: Improved resolution of high molecular mass proteins in two-dimensional gel electrophoresis

Paola Roncada

Paola Roncada

Istituto Sperimentale Italiano, L. Spallanzani, Milan, Italy

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Marina Cretich

Marina Cretich

Istituto di Chimica del Riconoscimento Molecolare, C.N.R., Milan, Italy

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Riccardo Fortin

Riccardo Fortin

Istituto Sperimentale Italiano, L. Spallanzani, Milan, Italy

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Susanna Agosti

Susanna Agosti

Istituto di Chimica del Riconoscimento Molecolare, C.N.R., Milan, Italy

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Lucia De Franceschi

Lucia De Franceschi

Dipartimento di Medicina Clinica e Sperimentale Università di Verona, Italy

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Gian Franco Greppi

Gian Franco Greppi

Istituto Sperimentale Italiano, L. Spallanzani, Milan, Italy

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Francesco Turrini

Francesco Turrini

Porto Conte Ricerche, Alghero (SS), Italy

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Franco Carta

Franco Carta

Porto Conte Ricerche, Alghero (SS), Italy

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Stefano Turri

Stefano Turri

Department of Chemistry, Materials and Chemical Engineering “Giulio Natta”, Polytechnic of Milan, Italy

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Marinella Levi

Marinella Levi

Department of Chemistry, Materials and Chemical Engineering “Giulio Natta”, Polytechnic of Milan, Italy

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Marcella Chiari

Corresponding Author

Marcella Chiari

Istituto di Chimica del Riconoscimento Molecolare, C.N.R., Milan, Italy

Istituto di Chimica del Riconoscimento Molecolare C.N.R., Via Mario Bianco 9, I-20131 Milano Italy Fax: +39-022-8500-035===Search for more papers by this author
First published: 02 June 2005
Citations: 14

Abstract

A method was developed in order to analyse high molecular mass proteins by two-dimensional (2-D) electrophoresis using a copolymer of acrylamide and allyl agarose instead of Bis cross-linked polyacrylamide (PA) gels in sodium dodecyl sulphate-electrophoresis. In this work, the matrix composition was optimised to improve the resolution of proteins larger than 200 kDa. The new gel type does not entrap large proteins and protein complexes at the application site. Mechanical properties were investigated through rheological measurements, which suggested the formation of a highly entangled elastomeric soft gel. A high 2-D resolution of proteins, extracted from membranes of red blood cells, was obtained in these gels. An example of tryptic digestion, peptide extraction and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry was reported. The results demonstrate that the new gel is fully compatible with mass spectrometry protein analysis.