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Aim: To establish and optimize two-dimensional electrophoresis (2DE) and relevant techniques for the study of serum proteome of the patients with uremia, and compare serum protein 2DE pattern between the uremia patients and the normal. Methods: Using immobiline pH gradients isoelectricfocusing (IEF) as the 1st dimension and vertical SDS-PAGE as the 2nd dimension, we performed the comparison and conditional optimization of the factors. Immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis,silver staining, ImageMaster 2D 5.0 analysis software, matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-TOF-MS) and IPI human database searching, were used to separate and indentify the proteome of the sera from the patients with uremia. Results: Satisfactory 2DE patterns of the serum proteins were obtained. 26 protein spots were remarkably hanged in uremia patients, and 20 protein spots were identified by MALDI-TOF-TOF-MS.. Conclusion: Good reproducibility could be obtained by applying immobilized pH gradient 2DE to separate and identify the proteome in sera, which contributes to the further study on uremia toxins pertaining to protein.