Protein extraction from rice (Oryza sativa L.) root for two-dimensional electrophresis

Abstract

An efficient protein extraction method for two-dimensional gel electrophoresis (2-DE) from plant samples is usually challenging due to the low protein content and high level of interfering compounds. Proteomic analyses of rice (Oryza sativa L.) roots are limited by the lack of an efficient protein extraction method. To establish an effective protocol of protein extraction suitable for 2-DE analysis in rice roots, we evaluated three protein extraction methods (trichloroacetic acid [TCA]/acetone, Mg/NP-40/TCA, and tris-base/acetone). Our results showed that the Mg/NP-40/TCA extraction method had the highest protein yield and is the best resolution of protein separation among the three methods. The TCA/acetone method exhibited clear €protein€ profiles€ and €detected€ more €protein €spots with the highest intensity in the region of high Mr (above 45 kDa) than the other methods. However, this method was unable to detect proteins with low-Mr (less than 24.0 kDa). The Tris-base/acetone method showed the poorest resolution of protein separation. Our results suggest that the Mg/NP-40/TCA method was the most effective among the three methods and may provide enhanced proteomic information for rice and other crop roots.

Key words： protein extraction; two-dimensional electrophresis (2-DE); rice (Oryza sativa L.) roots

References

1 Bradford M M (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 72(1-2): 248-254

2 Candiano G, Bruschi M, Musante L, Santucci L, Ghiggeri G M, Carnemolla B, Zardi L, Righetti P G (2004). Blue silver: A very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis, 25(9): 1327-1333

3 Damerval C, De Vienne D, Zivy M, Thiellement H (1986). Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat-seedling proteins. Electrophoresis, 7(1): 52-54

4 Görg A, Weiss W, Dunn M J (2004). Current two-dimensional electrophoresis technology for proteomics. Proteomics, 4(12): 3665-3685

5 Hille J M, Freed A L, Wätzig H (2001). Possibilities to improve automation, speed and precision of proteome analysis: a comparison of two-dimensional electrophoresis and alternatives. Electrophoresis, 22(19): 4035-4052

6 Kim S T, Cho K S, Jang Y S, Kang K Y (2001). Two-dimensional electrophoretic analysis of rice proteins by polyethylene glycol fractionation for protein arrays. Electrophoresis, 22(10): 2103-2109

7 Laemmli U K (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259): 680-685

8 O’Farrell P H (1975). High resolution two-dimensional electrophoresis of proteins. J Biol Chem, 250(10): 4007-4021

9 Rabilloud T (1998). Use of thiourea to increase the solubility of membrane proteins in two-dimensional electrophoresis. Electrophoresis, 19(5): 758-760

10 Shaw M M, Riederer B M (2003). Sample preparation for two-dimensional gel electrophoresis. Proteomics, 3(8): 1408-1417

11 Xie C J, Wang D, Yang X Y (2009). Protein Extraction Methods Compatible with Proteomic Analysis for the Cotton Seedling. Crop Science Society of America, 49(2): 395-402

12 Xu C P, Xu Y, Huang B R (2008). Protein Extraction for Two-Dimensional Gel Electrophoresis of Proteomic Profiling in Turfgrass. Crop Science Society of America, 48(4): 1608-1614

13 Yang Q S, Wang Y Q, Zhang J J, Shi W P, Qian C M, Peng X X (2007). Identification of aluminum-responsive proteins in rice roots by a proteomic approach: cysteine synthase as a key player in Al response. Proteomics, 7(5): 737-749