THE UNIVERSITY OF MANITOBA STUDIES ON OVINE PLACINTAL ...

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Pur lf icat,ion Procedures
AII stepF v¡ere carrj.ed out at 4 C unless otherr¡ùise
specified.
1. Extraction 3
Placenta1 t.issues which were obtained at the time
of surgery or slaughter v¡ere immediat,ely frozen at -20 C with.
out separating mat.ernal and foetal cotyledons. At the time
of extract,ion, approximateLy 9-lO kS(2O-.22 lbs) of placentaL
cotyledons hrere homogenized wíth a Polytron pT-L0 (Brinkmann)
homogenízer at. maximum speed for 30-60 seconds in 0.05M glycine-
hydroxide buffer, adjûsted to pH 9.5 with I N ammonium
hydroxide, using a ratio of buffer to tissue of 5:1 (v,/w).
The homogenate rdas stirred overnight and then centrifuged at
20,000 x g for 20 min. The petLet was discarded.
2. Ácidification!
To the supernatant. h¡as added slowly glacial acetic
acid to a final pH of 6.5 with constant st.irring. After alldsr-
ing the precipitate to settle overnight, the mixture was
centrifuged at. 20,000 x g for 20 min. , the precipitate vras
discarded . To the supernatant was added slo!,rly
ammonium hydroxide to achieve a final plt of 9.0.
3. Anion Exchange Chromatography:
2N
The pH 9.0 solution hras diluted with an equaì vo.lume of
distilled b¡ater and was applied to a column ( 15 x 50 cm )
of diethylarninoethyl cellulose (Whatman DE-32) previously
equilibratecl with 0.01 M glycine-hydroxide buffer, pH 9.0.
After thej colurnn tras vra9hed hrith 3 to 4 bed..yolume of, starÈing