title: Reversed-phase high-performance liquid chromatography method for the determination of prolactin in bacterial extracts and in its purified form.
abstract: Reversed-phase high-performance liquid chromatography methodology for the determination of human prolactin (hPRL) in bacterial periplasmic space or in purified preparations has been developed. The technique, based on the high hydrophobicity of the hPRL molecule, allows its separation from the bulk of bacterial proteins. The precision for periplasmic shock fluid analysis was characterized by relative standard variations of 3-7% for intra-day and of 3-25% for inter-day determinations. Accuracy, evaluated by recovery tests, was of the order of 90%, a calibration curve being constructed with the use of a lyophilized osmotic shock fluid extract, which provided a stable, readily prepared internal reference. Sensitivity was of the order of 0.5 microg of hPRL. The methodology developed also provided a tool for comparing the hydrophobicity of glycosylated and non-glycosylated prolactin molecules obtained from several different species and of different preparations of native or biosynthetic human prolactin.
authors.affiliation: Biotechnology Department, IPEN-CNEN, Cidade Universitária, São Paulo, Brazil.
authors.forename: Carlos R J
authors.initials: CR
authors.lastname: Soares
authors.forename: Iara M C
authors.initials: IM
authors.lastname: Camargo
authors.forename: Ligia
authors.initials: L
authors.lastname: Morganti
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Gimbo
authors.forename: João
authors.initials: J
authors.lastname: Ezequiel de Oliveira
authors.forename: Richard
authors.initials: R
authors.lastname: Legoux
authors.forename: Pascual
authors.initials: P
authors.lastname: Ferrara
authors.forename: Paolo
authors.initials: P
authors.lastname: Bartolini
mesh_terms: D001419:Bacteria / Q000737:chemistry*; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D011388:Prolactin / Q000032:analysis* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000032:analysis / Q000302:isolation & purification; D015203:Reproducibility of Results
chemical_list: D011994:Recombinant Proteins; D011388:Prolactin