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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
Author(s) -
Bustamante Juan J.,
Garcia Macario,
Gonzalez Leticia,
Garcia Jesus,
Flores Rafael,
Aguilar Roberto M.,
Trevino Ana,
Benavides Luis,
Martinez Andrew O.,
Haro Luis S.
Publication year - 2005
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200500439
Subject(s) - molecular mass , chromatography , polyacrylamide gel electrophoresis , chemistry , gel electrophoresis , electrophoresis , molecular weight size marker , elution , two dimensional gel electrophoresis , gel electrophoresis of proteins , biochemistry , enzyme , proteomics , gene
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.