Evaluation of an effective sample prefractionation method for the proteome analysis of breast cancer tissue using narrow range two-dimensional gel electrophoresis.
One method of improving the protein profiling of complex mammalian proteomes is the use of prefractionation followed by application of narrow pH range two dimensional (2-D) gels. The success of this strategy relies on sample solubilization; poor solubilization has been associated with missing protein fractions and diffuse, streaked, and/or trailing protein spots. In this study, I sought to optimize the solubilization of prefractionated human cancer cell samples using isoelectric focusing (IEF) rehydration buffers containing a variety of commercially available reducing agents, detergents, chaotropes, and carrier ampholytes. The solubilized proteins were resolved on 2-D gels and compared. Among five tested IEF rehydration buffers, those containing 3-[(3-cholamidopropyl)dimethylamino]-1-propane sulfonate (CHAPS) and dithiothreitol (DTT) provided superior resolution, while that containing Nonidet P-40 (NP-40) did not significantly affect protein resolution, and the tributyl phosphine (TBP)-containing buffer yielded consistently poor results. In addition, I found that buffers containing typically high urea and ampholyte levels generated sharper 2-D gels. Using these optimized conditions, I was able to apply 2-D gel analysis successfully to fractionated proteins from human breast cancer tissue MCF-7, across a pH range of 4-6.7.
Lee
KiBeom
K
Department of Biotechnology, SongDo Techno Park, 7-50 Songdo-Dong, Yeonsu-Gu, Incheon 406-840, Republic of Korea. klee02@empal.com
D001943:Breast Neoplasms / Q000378:metabolism*; D002021:Buffers; D045744:Cell Line, Tumor; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D020543:Proteome / Q000032:analysis* / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D014867:Water
D002021:Buffers; D020543:Proteome; D014867:Water