Does anyone have experience with the assay of estrogen receptor? We are
trying to measure the binding capacity of estrogen
receptor found in fish liver. We would also like to do competitive
binding study with several potential xenoestrogens.
The assay method used H3-estradiol as the ligand,
diethylstylbestrol (100 X in excess) to discriminate for specific binding
and Dextran-Activated charcoal preparation to separate estradiol-receptor
complexes from free estradiol in the incubation mixture. Those familiar
with the Scatchart plot determination of estrogen receptor will
recognize the method.
We have been trying to measure the amount of estrogen receptor by
this technique which looks fairly simple but without
success for several week now
As a positive control (fish from which estrogen receptor should be at
their maximum) we used female trout at a stage representing about one month
before spawning. So we are pretty sure the receptor are there. We tried
several modifications to the technique of isolation and that of binding
assay (pH of buffer, concentration of glycerol etc)
If anyone out there knows of limitations and/or precautions regarding
this technique, we will greatly appreciate hearing from you.
Thanks in advance,
Pierre
