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Medical laboratory centre of expertise on Congenital Disorders of Glycosylation and Glycosylated ferritin
2-DE Western-blotting analysis
Two-dimensional electrophoresis (2-DE) of proteins from 2 µL of 10-fold diluted plasma is conducted using ZOOM strip pH 4-7 for the first dimension and ready-made 4-12 % NuPAGE Bis-Tris gel for the second dimension as recommended by the manufacturer (Life technologies). After 2-DE, proteins are transferred to nitrocellulose sheets and glycoforms of interest are both revealed using a mix of rabbit primary antibodies, HRP-linked anti-rabbit IgG secondary antibody (1/5000 v/v; GE Healthcare) and Chemidoc XRS (Bio-Rad).
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