Database: PubMed
Entry: 9504818
LinkDB: 9504818
Original site: 9504818 
     Reymond MA, Sanchez JC, Hughes GJ, Gunther K, Riese J, Tortola S,
     Peinado MA, Kirchner T, Hohenberger W, Hochstrasser DF, Kockerling F.
     Standardized characterization of gene expression in human colorectal epithelium 
     by two-dimensional electrophoresis.
     Electrophoresis. 1997 Dec;18(15):2842-8. doi: 10.1002/elps.1150181520.
     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 
     ( Some of these patterns might provide diagnostic or 
     prognostic markers, or even molecular targets for therapy in the future.

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