Partial purification and preparation of polyclonal antibodies against candidate chromatin acceptor proteins for the avian oviduct progesterone receptor.
Steroid hormones bind to specific receptors in target cells that in turn bind to chromatin acceptor sites to alter gene expression. These chromatin acceptor sites, for a variety of steroid receptors, appear to be composed of acceptor proteins tightly bound to the DNA. This paper describes the preparation of new polyclonal antibodies against the chromatin acceptor proteins of the avian oviduct progesterone receptor (PR) and their use in monitoring the purification of the acceptor proteins. This laboratory recently reported the preparation of monoclonal antibodies that do recognize the intact chromatin acceptor sites containing DNA-bound acceptor proteins but not the unbound acceptor protein for PR [Goldberger, A., Horton, M., Katzmann, J., & Spelsberg, T. C. (1987) Biochemistry 26, 5811-5816]. In order to obtain antibodies that recognize the unbound acceptor protein, polyclonal antibodies were prepared against a highly purified preparation of the acceptor protein(s). Analyses by ELISA indicate that the polyclonal antibodies recognize both the intact acceptor sites and the unbound (free) acceptor protein(s). Using these antibodies in Western immunoblots, two antigenic species of 10 and 6 kDa were detected in crude fractions of acceptor protein. These two protein species could be separated and further enriched while still retaining acceptor activity, i.e., the capacity to generate specific binding of the PR. Thus, the antigenic activity is closely associated with, if not identical with, the acceptor activity. Whether one or both species are used in vivo or whether the 6-kDa species is a proteolytic product of the 10-kDa species is unknown.(ABSTRACT TRUNCATED AT 250 WORDS)
Goldberger
A
A
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905.
Spelsberg
T C
TC
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D000936:Antigen-Antibody Complex; D002645:Chickens; D002843:Chromatin / Q000378:metabolism*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D008970:Molecular Weight; D010057:Oviducts / Q000378:metabolism*; D011980:Receptors, Progesterone / Q000235:genetics / Q000378:metabolism*
D000906:Antibodies; D000936:Antigen-Antibody Complex; D002843:Chromatin; D011980:Receptors, Progesterone