TY - JOUR
T1 - Specific immunoprecipitation method for isolating isoforms of insulin-like growth factor binding protein-3 from serum
AU - Bons, J.A.
AU - Michielsen, E.C.
AU - de Boer, D.
AU - Bouwman, F.G.
AU - Jaeken, J.
AU - van Dieijen-Visser, M.P.
AU - Rubio-Gozalbo, M.E.
AU - Wodzig, W.K.
PY - 2008/1/1
Y1 - 2008/1/1
N2 - BACKGROUND: This report describes an in-house developed immunoprecipitation method to isolate insulin-like growth factor binding protein-3 (IGFBP-3) and its isoforms from serum. The method was compared to other existing immunoprecipitation methods. The study of IGFBP-3 isoforms is relevant for further studies on congenital defects in glycosylation (CDG), galactosemia, and alcoholic liver cirrhosis. METHODS: Monoclonal and/or polyclonal anti-human IGFBP-3 antibodies were covalently immobilised on protein-A Sepharose beads using dimethyl pimelimidate as cross-linker. By incubation with these immobilised antibodies, intact IGFBP-3 and fragments of IGFBP-3 were isolated from serum. Enzyme-linked immunosorbent assay (ELISA) and one-dimensional gel electrophoresis (1-DE) experiments were performed to define the optimal immunoprecipitation method. Isolated proteins were separated by 1-DE and two-dimensional gel electrophoresis (2-DE) and visualised by Western blotting. RESULTS: ELISA and 1-DE results illustrated that an optimal isolation was performed using PBS for the incubation with serum. Laemmli sample buffer, containing 2-amino-2-(hydroxymethyl)-1,3-propanediol hydrochloride and sodium dodecyl sulfate, or urea/CHAPS was optimal for the elution. Clinical validation was performed using CDG-Ia serum samples. The 2-DE experiments showed characteristic isoform patterns for CDG-Ia. CONCLUSIONS: The optimized in-house developed immunoprecipitation method resulted in specific detection of IGFBP-3 isoforms and is suitable for further studies on glycosylation defects.
AB - BACKGROUND: This report describes an in-house developed immunoprecipitation method to isolate insulin-like growth factor binding protein-3 (IGFBP-3) and its isoforms from serum. The method was compared to other existing immunoprecipitation methods. The study of IGFBP-3 isoforms is relevant for further studies on congenital defects in glycosylation (CDG), galactosemia, and alcoholic liver cirrhosis. METHODS: Monoclonal and/or polyclonal anti-human IGFBP-3 antibodies were covalently immobilised on protein-A Sepharose beads using dimethyl pimelimidate as cross-linker. By incubation with these immobilised antibodies, intact IGFBP-3 and fragments of IGFBP-3 were isolated from serum. Enzyme-linked immunosorbent assay (ELISA) and one-dimensional gel electrophoresis (1-DE) experiments were performed to define the optimal immunoprecipitation method. Isolated proteins were separated by 1-DE and two-dimensional gel electrophoresis (2-DE) and visualised by Western blotting. RESULTS: ELISA and 1-DE results illustrated that an optimal isolation was performed using PBS for the incubation with serum. Laemmli sample buffer, containing 2-amino-2-(hydroxymethyl)-1,3-propanediol hydrochloride and sodium dodecyl sulfate, or urea/CHAPS was optimal for the elution. Clinical validation was performed using CDG-Ia serum samples. The 2-DE experiments showed characteristic isoform patterns for CDG-Ia. CONCLUSIONS: The optimized in-house developed immunoprecipitation method resulted in specific detection of IGFBP-3 isoforms and is suitable for further studies on glycosylation defects.
U2 - 10.1016/j.cca.2007.09.002
DO - 10.1016/j.cca.2007.09.002
M3 - Article
C2 - 17904539
SN - 0009-8981
VL - 387
SP - 59
EP - 65
JO - Clinica Chimica Acta
JF - Clinica Chimica Acta
IS - 1-2
ER -