Objective:To establish a two-dimensional gel electrophoresis(2-DE) protocol for theproteomic study of Hippocampus trimaculatus

in order to lay the foundation for further research on Hippocampus in the late stage. Method:The methods of protein extraction

lysis

purification

and sample loading quantity were optimized. Result:The optimized protein extraction method was the liquid nitrogen grinding method. The best lysate contained 7 mol·L-1 Urea

2 mol·L-1 Thiourea

4% Chaps

1 mmol·L-1 PMSF

65 mmol·L-1 DTT and 0.2% Bio-Lyte. The best protein purification method was ultrafiltration+TCA/acetone precipitation.The best sample loading quantity was 300 μg.A total of 236 protein spots were detected by image analysis

all of which were distributed within the range between 14.4-97.4 kDa

with unobvious differences. Protein isoelectric pointswere mainly distributed between pH 4-9

among which alkaline protein was the most abundant. Conclusion:The two-dimensional electrophoretic map obtained by the method mentioned above shows clear spots

uniform separation and a good quality. The method is suitable for the proteomic analysis of H. trimaculatus. This study can lay the foundation for further studying the proteomics of H. trimaculatus and screening characteristic proteins.