Sample preparation is one of the major issues in 2-DE for the separation of proteins. Although a 100% representation of cellular proteins onto a 2-DE is virtually impossible, maximum representation of cellular proteins compared with the original cell lysate is important in the subsequent analysis. We demonstrate that lysis of cells in urea/thiourea solution with subsequent sonication to disrupt the nucleic acids and concentration of the lysate using centri-con led to enrichment of proteins. The procedure resulted in minimal nucleic acid contamination with better resolution of spots. 2-DE spot patterns of proteins prepared using urea - thiourea solubilization/centri-con method to other protein enrichment methods such as phenol/chloroform/isoamyl alcohol extraction, methanol/ammonium acetate precipitation, acetone precipitation and ethanol precipitation were compared. Urea - thiourea solubilization combined with centri-con method of protein enrichment represented higher number/unique spots particularly in the 50-250 kDa M r compared with others. Lysis of cells in urea/thiourea from the beginning of lysate preparation preserves the proteins from protease activity due to denaturation of proteases. Thus, we demonstrate that the centri-con methodology is simple and effective for the preparation of high-quality sample that can be used for a qualitative representation of cellular proteins on a 2-DE for proteomic analysis. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.
cited By (since 1996)2
T. Xavier, ,, Ganesan, T. S., and Menon, K. N., “A simple and efficient method for processing of cell lysates for two-dimensional gel electrophoresis”, Electrophoresis, vol. 31, pp. 2429-2435, 2010.