THE UNIVERSITY OF MANITOBA STUDIES ON OVINE PLACINTAL ...

103
B : MFTÍÍOD OF DETECTION AND CHARACTERIZATION OF THE
RFCE?TOR BTNDING STTTES FOR OVINE PLACENTAL LACTO_
GEN IN THE SflEEP
MATERTALS AND METHODS
Tissue Receptor PreÞarat.ion
Sheep tissue were obtained within 5 min of the death
of the animaLs and were immecliately frozen at -20 C until
required. All tissues were processed within 6 months. At the
tilne of preparation of 1O0r0O0 x g mícrosomal fractions, tissues
were thalved and honogenízed in ice cold 0.3M sucrose i¡i a
polytron PT-l-o (Brinkmann Instruments, Inc., Westbury. N.y.)
homogenizer set at maximum speed for 30-60 sec, except for
the adipose tissues which were homogenized lrith 0.3M sucrose
at 22C" The ratio of tissue to sucrose was 1¡5 (wt,/voL).
After homogenízation, centrifugatíon was carried out as
described previously (Shiu et al ,1973). Initiallyr the hcirmo-
genate was centrifuged at 31000 rpm for 30 min. The
supernatnant v¡as separated from the pe11et(TB0 x g fract.íon)
and subjected to a second. period of centrifugation at 11,500
rpm for 20 min. Again the supernat,ant was separated from the
peJ-let(15,000 x g fraction) and hras subjected to final centri-
fugation at 451000 rpm fo,:r 90 min. The pellet obtained at Èhe
final stage was cal-Ied the 1001000 x g microsomal petlet.
The pellet was guspended in 0.025M Tris-HCl , pH 7.6, contain-
ing 10 nM MgCl2 and stored at, -20C until use"