THE UNIVERSITY OF MANITOBA STUDIES ON OVINE PLACINTAL ...

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buffer, the eluates were pooled, The naterials bound to the
colunn were discarded.
4. Cation Exchange Chromatography 3
The pooled eluate was acidified stowly with glacial
äcetic acid, to a final pH of, 6.0 . After stirring the sotrution
overnightf the solution was centrifuged at Zg0 x g
f,or 20 min. The precipitate was discarded.
The supernatant was applied to a colunn ( 15 x 50 cm)
of carboxynethyL celtulose ( Whatman CM-32 ) which was also
equilibrated with 0.01 M anunoniurn acetate buffer, pH 5.5.
After washing the column with 20 Liters of starting buffer,
0.01 M anunonium acetate, pH 5.5, the colúmn wâs eluted with
a stepwise NaCl grâdient (0.05f. 0.I, O.Z, and 0.5Mi . ttie :: .
fractions containing oPL were pooled .,:.r ,
5. Carboxylmethyl- Sephadex colu¡trd chromatography:
The pooled fractions were further diluÈed with 3
eeual y6l¡¡s5 of distilled water, and applied to a column of
carboxl¡methyl-sephadex ( CM-sephadex C-50rPharmacia; 6 x 15
cm) which r4ras equilibrated r.¡Íth 0. 01 M sodiu¡n acetate, pH 6. 0.
After the colunn was washed with 5 bed volumes of 0.01M
sodium acetate buffer, pH 6.0 containing 0.05M NaCl . a linear
NaCI gradient $ras begun using 0.01M sodium acetate, pH 6.0
containing. 0.05M sodium chloride as the initial buffer and
0.01M sodium acetate, pH 6.0 containing 0.5M NaCl as Èhe
limiting buffer. The fractions containíng opl., ldere pooled
and concentrated to a Ê¡ûa1l volume b)¡ ultrafittration by
using an Amicon membrane UM10.