A microscale protocol for the isolation of transferrin directly from serum

Clinica Chimica Acta; International Journal of Clinical Chemistry
Ana PenezićOlgica Nedić

Abstract

A microscale procedure for the isolation of transferrin directly from human serum (hTf) is described in this study. The protocol is based on three precipitation steps without application of chromatography. It lasts 90min with the initial sample volume of 250μL. The yield of the isolated hTf is 58%, which is considerable in biochemical terms. The purity of the isolated hTf is 97%, as assessed by three methods: electrophoresis followed by protein staining, immunoblotting and HPLC. Immunoblotting with antibodies against other major serum proteins indicated that isolated hTf does not contain albumin, immunoglobulin G or alpha-2-macroglobulin. Lectin dot-blot demonstrated that isolated hTf preserved its glycan moieties. Fluorescent emission spectroscopy of the isolated hTf has shown no changes in tertiary structure. Isolated hTf was approximately 26% saturated with iron ion, which is comparable to physiological value (although a degree of saturation decreases to some extent during isolation procedure). Finally, co-immunoprecipitation experiment confirmed that isolated hTf retained its ligand characteristics crucial for the ligand-receptor type of interaction with the hTf receptor. To conclude, the procedure described in this work, i...Continue Reading

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Citations

May 6, 2019·Proteomics. Clinical Applications·Ana PenezićOlgica Nedić
Mar 28, 2020·Critical Reviews in Analytical Chemistry·Mariza Aires FernandesMarlus Chorilli

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