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Protein Extraction for Two-Dimensional Gel Electrophoresis of Proteomic Profiling in Turfgrass

Dep. of Plant Biology and Pathology, Rutgers Univ., New Brunswick, NJ 08901

^* Corresponding author (huang{at}aesop.rutgers.edu).

Protein extraction for two-dimensional gel electrophoresis (2-DE) from plant samples is challenging due to low protein content and high level of contaminants. Proteomic research in turfgrass is limited by the lack of efficient protein extraction methods. To establish an effective protocol of protein extraction suitable for 2-DE analysis in turfgrasses, four protein extraction methods (chloroform/acetone, tris-base/acetone, phenol/ammonium acetate, and trichloroacetic acid [TCA]/acetone methods) were evaluated for creeping bentgrass (Agrostis stolonifera L.). Proteins were extracted from leaves and roots of mature plants using the above four methods and separated by 2-DE. The TCA/acetone extraction had higher protein yield and resolution of protein separation for leaves, compared to the other three methods. For roots, both the TCA and phenol methods had higher protein yields and number of protein spots than the other two methods. The phenol method had more protein spots and higher spot intensities in the low molecular weight (M_r) region or high isoelectric point (pI) region than the TCA extraction, while more protein spots and higher spot intensity in the region of high M_r were detected by the TCA method than by the phenol method. Our results suggested that the TCA method was the most effective among the four methods for leaves, and a combination of the TCA and phenol methods may provide enhanced proteomic information for roots in turfgrass.

Abbreviations: 2-DE, two-dimensional gel electrophoresis • CHAPS, 3-[(3-cholamidopropyl)dimethylamonio]-1-propanesulfonate • DTT, dithiothreitol • IEF, isoelectric focusing • IPG, immobilized pH gradient • M_r, molecular weight • PAGE, polyacrylamide gel electrophoresis • pI, isoelectric point • TCA, trichloroacetic acid

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Received for publication November 15, 2007.