THE UNIVERSITY OF MANITOBA STUDIES ON OVINE PLACINTAL ...

Char4ctêf ization Pfocedures
l. Chêmícal
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(a) Analytical ge1 electrophoresis ¡
Polyacrylamide geI electrophoresis was carried out
as described by Davis (1964) and Reisfeld et aL (1962) r{íth
s1ight, modification. In alkaline gel, 7.2* acrylamide and
pH 9.5 were used., whereas under ácidic condition, 9.Ot
acrylamide and pH 4.3-4.5 were used.. In both conditions,
duplicate samples ltlere run . One gel was stained with dye
while the other $¡as cut serially and the individual segments
were eluted in l mI of 0.1M Tris-HCIr pH 7.6 containing 0.1t
BSA at 4 C fot 24 hours wíth shaking. The eluants subsequently
were analyzed by the radioreceptoras say for growth hormone.
The staining was performed by placing the gel ín l€l
Amido Black dye ( in 78 acetíc acid) for L or 2 hours. The
stained gel was removed, from the staining solution and placed
in a test tube with 78 acetic acid to destain until distinct,
bands were seen.
For the alkaline gel-, bromophenol blue was used as
a tracking dye, whereas for the acidic ge1, Basíc Fuchsin
dye was used.
(b) Analytical gel isoeLectrictfocusing electrophoresis:
Analytical thin layer polyacrylanide geJ. Ísoelectric-
focusing was perforrned using a LKB 21L7 multiphor apparatus.
Focusing v¡as achieye¿l using a pH range 3.5 to 9.5 t¡ith a 6t
(w/y) concentration of acryLamide and a crossFlinking of 2.5t.