A polyclonal antibody to the rat oestrogen receptor expressed in Escherichia coli: characterization and application to immunohistochemistry.
A rat oestrogen receptor-beta-galactosidase fusion protein was expressed using a pEX2/rat oestrogen receptor cDNA construct. Scatchard analysis of [3H]oestradiol-17 beta binding to the cell lysate revealed that the fusion protein had functional binding sites specific for oestradiol with a dissociation constant of 1.49 nmol/l. The relative molecular weight (M(r)) of the fusion protein was determined as 180,000 by immunoblot analysis of the cell lysate employing a monoclonal antibody to the human oestrogen receptor. The protein was isolated by means of SDS-PAGE and subsequent electroblotting. By immunization with the purified materials on nitrocellulose membrane, a polyclonal antibody to the rat oestrogen receptor was raised in a rabbit. Binding of [3H]oestradiol to the oestrogen receptor from the rat uterus was inhibited by the antibody in a dose-dependent manner. The antibody was also able to recognize the oestrogen receptor occupied by [3H]oestradiol. Thus, the antibody could react with both forms of the receptor molecule, either occupied or unoccupied by the hormone. In immunoblot analysis of the cytosol fraction of the rat uterus, a single band of M(r) 67,000, the size of the oestrogen receptor, was detected by the antibody. Moreover, when the antibody was applied to immunohistochemical examination of paraffin-embedded pituitary and brain sections of the rat, immunostaining was observed in cells of the anterior pituitary and in neurones in specific regions of the brain. The immunoreactivity was restricted exclusively to cell nuclei in both tissues.(ABSTRACT TRUNCATED AT 250 WORDS)
Okamura
H
H
Department of Anatomy and Embryology, Tokyo Metropolitan Institute for Neuroscience, Japan.
Yamamoto
K
K
Hayashi
S
S
Kuroiwa
A
A
Muramatsu
M
M
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D004926:Escherichia coli / Q000276:immunology*; D007150:Immunohistochemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D011960:Receptors, Estrogen / Q000276:immunology*
D000906:Antibodies; D011960:Receptors, Estrogen