THE UNIVERSITY OF MANITOBA STUDIES ON OVINE PLACINTAL ...

- 93-
(.TCA) solution were added consecutively to 0.1 nl gf the
dil-uted reaction mixture. After 3 'hours of incubation at.
4 C, the total raclioactívity of Èhe mixture rdas determined
and then the tube r¡7as centrifuged at, 780 x g for 20 min¡,
The supernatant $¡as decanted and the precipitate was counted.
in the LKB autogamma counter. The incopporat.ion of, the
radioact.ivity into protein is expressed as the TCA precipitable
radioactivity over the precount. of the TCA reaction
mixture as a percentage. The specific activity of the iodinated
hormone is defined as the total precipitable counts divided
by the amount of protein used for iodinaÈion(s ug). The per-
centage of incorporation into hcH t'7âs 55-75t, and its specific
activity was l-10-130 uCi,/ug of proteÍn.
3. Incubation procedure:
All dilutions were made with 0.025M Tris-HCI buffer,
pH 7.6 containÍng 0.1t BSA and L0 mM CacLz. The assays were
carriéd out in gJ.ass tubes ( L2X7 5 cm) containing 200 uI of
diluent, 100 ul of hormone standard (hcti), or IOO ul of knovùn
or unknown sanple, and L00 ut of [tr25r]-iodo hGH (approximat,ely
80,000 to 100,000 cpm). During the 3 hours incubation period
at room temperature, the tubes were shaken Vlgorously for
30 seconds every 30 minu The reaction was terminated by the
addition of 3 mI Íce-cold 0.025M sodium acetate,buf,fer, pH
5.4 containing 0.19 BSA, then centrifuged at, 780 x 9 for
20 min at 4 C. The supernatant was decanted and the nembrane
bound [125r]'.iodo hGH in the precipitate ldas counted in the
LKB autogallmA counter.