THE EXTRACTION AND PURIFICATION OF BOAR SPERM SURFACE PROTEIN
Keywords:
Immunological sexing, spermatozoa, sperm surface protein, sex sorted spermAbstract
Immunological sperm sexing is one of the desirable choices to separate X and Y sperm. The basicconcept of immunological technique is based on the different proteins on the surface of X and Y sperms.To investigate the sperm surface proteins, proteomic investigation using 2 dimensional gel (2D-gel)electrophoresis technique has been applied. The initial step, protein extraction is very important. Thus,the suitable strategies for sperm surface protein extraction from unsorted boar spermatozoa surfacewere considered for further use in 2D-gel analyses. The sperm surface proteins were extracted and thenpurified using Con-A Sepharose beads. After 22 h incubation, 2.625 mg/ml and 0.186 mg/ml of the totalprotein can be obtained using extraction solution with and without 0.5% Triton X-100, respectively.The small-sized proteins (~13-16 kDa) were the major products which can be extracted immediatelyin extraction solution containing Triton X-100. Almost all of the proteins, especially the small-sizedproducts (~13 and 16 kDa), were tightly bound to the Con-A Sepharose beads even in the presence of800 mM D-glucopyranoside. These results indicated that the major extracted proteins in Triton X-100solution were glycosylated proteins. In the future, surface proteins from sex sorted sperms (X spermor Y sperm only) will be extracted by this strategy and then the protein pattern on 2D-gels will becompared.
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