Abstract：In order to establish and optimize the two-dimensional electrophoresis (2-DE) system for testis proteins of matured male sheep (Ovis aries), different extracting methods, loading quantities, isoelectric focusing time and separation gel concentration were compared of their influence on the 2-DE maps. Results indicated that the best 2-DE maps were obtained through preparing proteins samples by the trichloroacetic acid(TCA)/acetone precipitation, loading 750 μg protein samples, isoelectric focusing with 75 000 Vh or more and 12% sodium dodecyl sulfonate (SDS) gel for protein separation. The optimized 2-DE system gives the maximum number of protein spots with clear background, which help to the study of male ovine testis development and male reproductive disease.
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