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Advanced liver disease can alter the levels of B12 binding proteins in the blood, making total serum B12 measurements unreliable—levels may appear normal or even elevated despite functional deficiency

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The PVDF membrane was subsequently washed (5 min × 5) with tris-buffered saline with tween (TBST) buffer and then incubated with the appropriate secondary antibody (Peroxidase-conjugated goat anti-rabbit IgG (H + L), at a dilution of 1:5000) in a 5% bovine serum albumin solution for 1 h at 25 °C