Proteome Analysis in BM2 Leukemia Cell Line: Determination of Optimal Conditions for Sample Preparation in Two-Dimensional Electrophoresis

  • P. Vana Department of Genetics and Molecular Biology, Faculty of Science, Masaryk University, Brno
  • J. Smarda Department of Genetics and Molecular Biology, Faculty of Science, Masaryk University, Brno

Abstrakt

Two-dimensional electrophoresis is a key experimental approach of proteomics. The quality of its results significantly depends on the method of sample preparation. The aim of this study was to set up optimal conditions for preparation of a sample of v-myb-transformed chicken monoblasts BM2 to obtain a high number of resolved proteins on two-dimensional gel. Starting compositions of both lysis buffer (pH 8 10.5) and rehydration buffer (pH 3 10) were modified by addition of thiourea causing a clear increase in the number of protein spots detectable on two-dimensional gel. In addition, sonication of the sample before its loading onto the gel further increased the number of resolved proteins as well as density of some of them. Addition of thiourea to the lysis and rehydration buffers and sonication increased the number of protein spots resolved in two-dimensional gel about eight-fold. The experimental conditions are used in detailed analysis of proteome changes during terminal differentiation of BM2 monoblasts. They can also be of interest for researchers using two-dimensional electrophoresis as a tool for proteome analysis of other leukemic cells.
Publikované
2005-01-15
Sekce
Články