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J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Apr 15;849(1-2):351-6. Epub 2006 Oct 10.

A general method for the extraction of citrus leaf proteins and separation by 2D electrophoresis: a follow up.

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  • 1National Research Council (CNR)-Institute of Biophysics (IBF), CNR Research Area, Via Moruzzi 1, 56124 Pisa, Italy. bianca.elena.maserti@pi.ibf.cnr.it

Abstract

With the aim of studying differentially expressed proteins as a function of abiotic and biotic stress in citrus plants, we optimized a protocol for the extraction of total leaf proteins and their 2-DE separation using commercially available immobilized pH gradient strips (IPGs) in the first dimension. Critical factors for good reproducibility of citrus leaf protein separation were identified: trichloroacetic acid (TCA)/acetone precipitation after extraction in lysis buffer, sample fractionation on narrow range overlapping IPGs and sample-cup loading at the anodic or cathodic end of the strip. The use of thiourea and a strong detergent (C7BzO) in the solubilization/rehydration buffer, coupled with the increase to 10% of SDS in the equilibration buffer before the second dimension seemed to affect positively the resolution of basic proteins. Using our protocol we resolved about 30 basic proteins on 6.3-8.3 pH range strips. Further, our protocol was successfully applied reproducibly on the analysis of control and salt exposed leaf samples of Citrus reshni Hort. Ex Tan.

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