title: Separation of proteins with a molecular mass difference of 2 kDa utilizing preparative double-inverted gradient polyacrylamide gel electrophoresis under nonreducing conditions: application to the isolation of 24 kDa human growth hormone.
abstract: A method for separating proteins with a molecular mass difference of 2 kDa using SDS-PAGE under nonreducing conditions is presented. A sample mixture containing several human growth hormone (hGH) isoforms was initially separated on a weak anion-exchange column. Fractions rich in 24 kDa hGH as determined by analytical SDS-PAGE were pooled and further separated by cation-exchange chromatography. The fractions pooled from the cation-exchange chromatography contained two hGH isoforms with a 2 kDa molecular mass difference according to SDS-PAGE analysis, 22 and 24 kDa hGH. The 22 and 24 kDa hGH were separated using continuous-elution preparative double-inverted gradient PAGE (PDG-PAGE) under nonreducing conditions. The preparative electrophoresis gel was composed of three stacked tubular polyacrylamide matrices, a 4% stacking gel, a 13-18% linear gradient gel, and a 15-10% linear inverted gradient gel. Fractions containing purified 24 kDa hGH were pooled and Western blot analysis displayed immunoreactivity to antihGH antibodies. PDG-PAGE provides researchers with an electrophoretic technique to preparatively purify proteins under nonreducing conditions with molecular mass differences of 2 kDa.
authors.affiliation: Department of Pathology & Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS, USA.
authors.forename: Juan J
authors.initials: JJ
authors.lastname: Bustamante
authors.forename: Macario
authors.initials: M
authors.lastname: Garcia
authors.forename: Leticia
authors.initials: L
authors.lastname: Gonzalez
authors.forename: Jesus
authors.initials: J
authors.lastname: Garcia
authors.forename: Rafael
authors.initials: R
authors.lastname: Flores
authors.forename: Roberto M
authors.initials: RM
authors.lastname: Aguilar
authors.forename: Ana
authors.initials: A
authors.lastname: Trevino
authors.forename: Luis
authors.initials: L
authors.lastname: Benavides
authors.forename: Andrew O
authors.initials: AO
authors.lastname: Martinez
authors.forename: Luis S
authors.initials: LS
authors.lastname: Haro
mesh_terms: D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D019382:Human Growth Hormone / Q000302:isolation & purification*; D006801:Humans; D008970:Molecular Weight; D010902:Pituitary Gland / Q000737:chemistry
chemical_list: D019382:Human Growth Hormone