THE UNIVERSITY OF MANITOBA STUDIES ON OVINE PLACINTAL ...

AFsa)¡- ProceiiureS
105
Specific binding studies were conducted hríth I000.i
50 ug protein, concentration being determined by the method
of lowry et al (1951). To each assay tube was addeil 0.2 m1 of
0.025M Tris-HCl buffer, pH 7.6 containing 0.1*BSA and L0 ¡nM
MgCI2t 0.I mI 100f000 x g pellet suspension, 0.1 m1 ovine
I125rlÍo¿o-PL (80 - l-o0,oo0 cpm) with or lvithout I ug of, opt,
in 0.l- rn1 of buffer, f,or a totaI. volume of 0.5 mI. The samples
wefe incubated at. 4 C for 24 hours. The reaction lsas terminated
by the addition of 3 mI ice-co'ld 0.025 M Tris-HCl buffer
containing 0.I* BSA and L0 nM MgC12. Bound and free hormone
were separated by centrifugation at 780 x g for 20 min at 4 C.
The supernatant ( free hor¡¡pne) was decanted by inverting the
tubes and allowing them to draj¡¡ for 30 min, and the membrane
bound ovine l125rl ioao-pL in the precipitate was deter¡nined
by counting the radioactivity in an LKB autoganma counter.
The percentage of specific binding of ovine t125rl-
iodo Pr to the tissue was calculated from the formula:
Specific binding = [ cpm bound to the tissue in the
Absence of unlabelled opL - cpm bound in the presence of
a Large excess (1 u9) of unlabeled oPL I x LOÙ/ total cpm
of ovine Ir25ll ioao-ÞL added
In the radioreceptorassay assay for oPL using various
oviine tissues (100,000 X g pellet), the assay procedures were
identícal with the above (_specif,ic bintting studies) sxcept
that ê known anount (yarious concentrations) of, unlabeLed
hormone or crude tÍssue extract hras added to the reaction