EXTRACTION AND PURIFICATION OF ZONA RADIATA PROTEINS (ZRPS) FROM BLUNTNOSE BLACK BREAM (MEGALOBRAMA AMBLYCEPHALA YIH)

This study deals with an effective procedure for extraction and purification of zona radiata proteins (Zrps) from eggshells of Bluntnose black bream (Megalobrama amblycephala Yih). To avoid harding resulting from parthenogenic activation, unfertilized eggs were immediately immersed in a large volume of ice cold buffer A. After homogenization and washing, the eggshells were frozen dry and solubilized in buffer B. The SDS-PAGE electrophoretic migration pattern shows the solubilized Zrps consiste mainly of three protein bands with the apparent molecular weight of 64KDa, 56KDa and 52KDa. To purify these three Zrps, the solubilized eggshell proteins were subjected to preparative SDS-PAGE. The protein bands were visualized using an ice-cold KCl solution. The opaque bands were excised from the gel and extracted using extraction buffer with electro-elution. SDS-PAGE pattern of purified Zrps shows highly purified Zrps were acquired and sufficient for further studies with immunoassay.