Bovine neurophysins I, II and C: new methods for their purification and for the production of specific antibodies.

1. Bovine neurophysins were prepared by a modified method, in which a Biogel P-60 column was used. This yielded two neurophysin fractions, the first containing neurophysin I and small quantities of the other neurophysins,the second containing neurophysin II and C, and only traces of neurophysin I. 2. Antibodies against neurophysin I, II and C were prepared by an original method, 5 mug in 100 mul water of each of the two fractions were applied on a gel slab and separated by iso-electric focusing in a pH gradient 4--6. The separated bands were visualized with 8-aniline-1-naphthalene sulfonic acid, magnesium salt and strips respectively containing neurophysin I, II or C were cut out. The neurophysin-containing strips were homogenized in complete Freund's adjuvant and injected into rabbits. 3. The specificity of the antisera were tested by immunocytochemistry and by radioimmunoassay. By this latter method, it was determined that cross-reactivity was less than 1%. The cross-reaction, observed with the immunohistochemical method could be eliminated by differential absorption. 4. It was found that neurophysin C antisera were undistinguishable from the neurophysin II antisera, while showing little cross-reactivity with the neurophysin I antisera. This suggests that in vivo neurophysin C is not a real neurophysin, or at least, that it is very similar to neurophysin II. 5. Highly purified bovic focusing method. By modifying a LKB Uniphor electrophoresis apparatus, the elute the proteins without switching off the voltage. The resolution of the technique is close to that offered by analytical gel iso-electric focusing.