THE UNIVERSITY OF MANITOBA STUDIES ON OVINE PLACINTAL ...

9B
6. Sephadex c-t00..gel fåltration i
The conceÍltrqted ÍlaterÍal was applied to a Sephadex
G-100 (Pharmacia, Uppsal-, Sweden) column (4.2 x I04 cm) which
was equilibrated v¡ith _0.01M glycine-hydroxide buffer, pH 10.0.
Fractions containing oPL hrere colLected and pooled.
7. Diethylaminoethyl--gephadex colurnn chromatography 3
The pooled fractj.ons were applied directly onto a
"oíot, (1 x 20 cm) of, diethylaminoethyl- sephadex (DE-sephadex
A-25¡ Pharmacia) which was equilibrated trith 0.01M glycine-
hydroxide buffer, pH 10.0. After the column was washed with
10 bed volumes of eluting buffer, 0.01M glycine-hydroxide
buffer, pH 10.0., a linear NaCl gradient was begun usíng
0,2M sodiun chloride as the final salt concentration. The
fract.ions containing oPt were collected, pooLed, and concent-
rated into a snal1 volume.
8. cel filtration:
Fina1ly, the concentrated material was apptied to a
colu¡nn (1.4 x 9 4 cm) of Sephadex G-50 (Superfine, pharmacia)
which v¡as equiLibrated with 0.05 M ar¡¡non ium bicarbonate,
pH I.2. The active fractions r,rere pooled, concentrated to a
volume of 2 to 3 ml , and then Lyophilized.