Standardized characterization of gene expression in human colorectal epithelium by two-dimensional electrophoresis
| Autor: | Miguel A. Peinado, Marc A. Reymond, Denis F. Hochstrasser, Graham J. Hughes, J Riese, Thomas Kirchner, Jean-Charles Sanchez, Klaus Günther, Ferdinand Köckerling, Silvia Tortola, Werner Hohenberger |
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| Rok vydání: | 1998 |
| Předmět: |
Time Factors
Clinical Biochemistry Biology Biochemistry Peptide Mapping Analytical Chemistry Dynabeads Intestinal mucosa Gene expression medicine Humans Electrophoresis Gel Two-Dimensional Intestinal Mucosa Polyacrylamide gel electrophoresis Epithelial Cells Cell sorting Reference Standards Molecular biology Epithelium Neoplasm Proteins Gene Expression Regulation Neoplastic medicine.anatomical_structure biology.protein Immobilized pH gradient Antibody Colorectal Neoplasms |
| Zdroj: | Electrophoresis. 18(15) |
| ISSN: | 0173-0835 |
| Popis: | New diagnostic and prognostic markers are needed in colorectal cancer. They can be found by differential analysis at DNA, RNA or protein level. The accuracy of phenotypic comparisons of tumor and normal tissues depends on the purity of the samples. We present an effective method to identify and isolate proteins that are differentially expressed under altered conditions, and a two-dimensional reference protein map of the normal human colonic epithelium. Normal colonic mucosa, primary tumors and liver metastases were prepared in the operating room. After washing in an ice-cold medium containing protease inhibitors, crypts were isolated by mechanical preparation without using metalloproteinases. Epithelial cells were then selected using Ber-EP4 Dynabeads. The samples were denaturated before processing for immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis according to SWISS-2DPAGE standards. The samples contained more than 95% epithelial cells as confirmed by fluorescence-activated cell sorting using pan-anticytokeratin antibodies. Cell surfaces were not damaged, as assessed by scanning electronic microscope. A protein reference map of the normal colonic epithelium was defined. Using gel matching, N-terminal sequencing and/or immunoblotting techniques, 60 polypeptides - including proteins specifically expressed in colorectal epithelium - have now been identified. This reproducible method of sample preparation permits the comparison of protein patterns found in various pathological states with the present reference map (http://www.expasy.ch). Some of these patterns might provide diagnostic or prognostic markers, or even molecular targets for therapy in the future. |
| Databáze: | OpenAIRE |
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