Isolation, characterization and localization of a lectin within the vitelline membrane of the hen’s egg
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Fig. 2. Rabbit polyclonal antibody to the purified lectin. In (A) Ouchterlony gel diffusion analysis was performed with rabbit antiserum (central well: pattern on the left-hand side obtained with unadsorbed serum and on the right-hand side the serum has been adsorbed with Type I cells) against vitelline membrane extract (c; 0·36 mg protein ml−1) and a solution of purified lectin concentrated with carbowax (p; 1·72 mg protein ml−1) together with solubilized blastoderms (b) and plasma membranes obtained from a suspension of blastoderm cells (m) and ovalbumin (ov) and ovomucoid (o). The precipitin pattern was stained with Amido black 10B. Rocket immunoelectrophoresis (B) was performed on purified lectin concentrated with carbowax (3·32 mg protein ml−1) and then serially diluted (from left to right) with PBS, 3 μl was placed in each well and antiserum (1 ml) was incorporated into agarose (12 ml) running gel. In this case the precipitin pattern was stained with Coomassie brilliant blue R-250. The rabbit anti-serum was shown by immunoblotting (C) to react with the subunit of MT 62000. Concentrated lectin solution (1· 72 mg protein ml− 1) was subjected to electrophoresis in an 8 % acrylamide gel before electrophoretic transfer to nitrocellulose paper. Antigen was visualized either by (1) immunoperoxidase staining using rabbit antiserum (1/10 dilution) and the ABC technique (Vectastain kit) in which biotinylated anti-rabbit immunoglobulin was the second antibody and diaminobenzidine tetrahydrochloride the peroxidase substrate, or by (2) autoradiography with 125I-labelled protein A (106 c.p.m. 3 mm− 1 wide strip). The strip on the right hand side (3) was stained with Coomassie brilliant blue R-250.