Extraction of rat pituitary prolactin for radioimmunoassay

The bulk of Prolactin (PRL) in rat pituitary gland stored as big molecular forms in secretory granules is mostly excluded from radioimmunoassay (RIA) determinations because secretory granules remain intact after tissue homogenization. Also big PRL is little immunoreactive and must be deaggregated to monomers to allow a complete detection by RIA. Different dissociating agents, used to render PRL monomers soluble, were tested at various temperatures and pH conditions. Incubations of pituitary homogenates in buffers at neutral pH yield consistent levels of PRL, but the sole alkalinization of the media increases significantly the radioimmunoassayable PRL content. No significant increase was detected with EDTA and thiols. The 2.5 M urea was the most effective extraction agent increasing about 7-fold the quantification of PRL by RIA. Extraction of PRL with urea was enhanced at pH 9.0 and at 4 C and this combination constitutes the method of choice for a complete extraction of pituitary PRL.