Establishment and optimization of twodimensional electrophoresis system for proteome analysis of haemolymph in Plutella xylostella

Key Words：twodimensional electrophoresis, proteomics, Plutella xylostella

Abstract：twodimensional electrophoresis system suitable for hemolymph proteome analysis in the diamondback moth, Plutella xylostella(L.), was developed. The effects of different experimental conditions; such as sample processing program, IEF time, staining methods, etc, on the results of twodimensional electrophoresis were analyzed and optimized. The results show that the TCA/acetone precipitation method had less protein loss, a clearer pattern, higher resolution and greater repeatability. Different lengths of gel strip needed different optimum amounts of sample protein and time; for strips 7, 17 and 24 cm in length the optimum amount of sample protein was 20-50 μg, 50-300 μg and 100-500 μg, respectively, and the appropriate focused time was 24 000 vhr, 50 000 vhr and 65 000 vhr, respectively. Strips were subjected to twodimensional electrophoresis after transfer onto 12% SDSpolyacrylamide gel in order to attain evenly distributed protein spots. Silver staining was significantly more sensitive than Coomassie brilliant blue, resulting in more protein spots being detected, however, Coomassie blue has the advantage that protein spots can be identified by mass spectrometry.