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《Journal of Ningbo University(Natural Science & Engineering Edition)》 2013-02
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Optimization of Protein Isolation and Method of Two-dimensional Electrophoresis from the Ink Sac in Sepiella maindroni

ZHAN Ping-ping1,2,WANG Chun-lin1,2,SONG Wei-wei1,2,MU Chang-kao1,2,LI Rong-hua1,2(1.School of Marine Science,Ningbo University,Ningbo 315211,China;2.Key Laboratory of Applied Marine Biotechnology of Ministry of Education,Ningbo University,Ningbo 315211,China)  
In order to develop an efficient protein extraction method suitable for two-dimensional electrophoresis(2-DE) from the ink sac in Sepiella maindroni,we compared four analytical approaches,which are the precipitation separation of TCA/acetone,the lyolysis buffer,the lyolysis-ultracentrifugation and the column chromatography for total protein extraction.We also compared the effects of different protein extraction methods,different IPG strips and different volumes of sample loaded on the 2-DE.The results show that the TCA/acetone precipitation is the most suitable method for the ink sac protein sample preparation for 2-DE,and the appropriate electrophoresis pattern can be obtained by loading 300 μg protein samples using the method of TCA/acetone precipitation,with IPG strips of pH 5-8 for the first dimensional electrophoresis and staining with silver nitrate.A total of(389±19) protein points are obtained by conducting image analysis.The improved TCA/acetone precipitation can achieve the highest resolution of two dimensional electrophoresis and high yield of protein,and the findings can facilitate the research of proteomics on the ink sac in Sepiella maindroni.
【Fund】: 国家自然科学基金(41176124 41206114);; 教育部博士点基金(20103305110003)
【CateGory Index】: S917.4
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