Optimization of Protein Extraction from Hepatopancreas of Litopenaeus vannamei and Effect of Environmental Stress during Waterless Transportation on Apoptosis-Related Proteins

In the present study, in order to improve the reliability of Western blotting results, the effects of extraction buffers, grinding methods, inhibitor type and concentration, and extraction time on protein extraction from the hepatopancreas of Litopenaeus vannamei were explored by considering protein dissolution rate and the completeness of protein bands in electrophoretic gels. Next, the expression levels of apoptosis-related proteins in the hepatopancreas of Litopenaeus vannamei were detected with Western blotting. The results showed that the protein dissolution rate was higher using RIPA lysis buffer compared with phosphate buffer saline (PBS) or water as the extraction solvent. Electric homogenization and liquid nitrogen grinding provided more complete protein bands. The degradation of proteins by endogenous proteases was obviously suppressed by addition of 4% protease and phosphatase inhibitor cocktail. In addition, Western blotting results revealed that in addition to inducing Litopenaeus vannamei into dormancy, low temperatures induced slight apoptosis in the hepatopancreas tissue. Furthermore, the apoptosis level increased with waterless duration and this effect was alleviated after removal of environmental stress.