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.
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.
Bustamante
Juan J
JJ
Department of Pathology & Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS, USA.
Garcia
Macario
M
Gonzalez
Leticia
L
Garcia
Jesus
J
Flores
Rafael
R
Aguilar
Roberto M
RM
Trevino
Ana
A
Benavides
Luis
L
Martinez
Andrew O
AO
Haro
Luis S
LS
D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D019382:Human Growth Hormone / Q000302:isolation & purification*; D006801:Humans; D008970:Molecular Weight; D010902:Pituitary Gland / Q000737:chemistry
D019382:Human Growth Hormone