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J Chromatogr B Analyt Technol Biomed Life Sci. 2017 Apr 15;1051:68-74. doi: 10.1016/j.jchromb.2017.03.010. Epub 2017 Mar 11.

Simplified purification approach of urinary neutrophil gelatinase-associated lipocalin by tangential flow filtration and ion exchange chromatography.

Author information

1
Division of Biochemistry, Department of Chemistry, Savitribai Phule Pune University, Ganeshkhind, Pune, 411007 Maharashtra, India; Laboratory of Native Proteins, Research and Development Division, Yashraj Biotechnology Ltd., Navi Mumbai, 400705 Maharashtra, India.
2
Laboratory of Native Proteins, Research and Development Division, Yashraj Biotechnology Ltd., Navi Mumbai, 400705 Maharashtra, India. Electronic address: sham83badgujar@gmail.com.
3
Laboratory of Native Proteins, Research and Development Division, Yashraj Biotechnology Ltd., Navi Mumbai, 400705 Maharashtra, India.
4
Division of Biochemistry, Department of Chemistry, Savitribai Phule Pune University, Ganeshkhind, Pune, 411007 Maharashtra, India.

Abstract

This investigation reports a simplified approach for the purification of urinary siderocalin known as neutrophil gelatinase-associated lipocalin (NGAL). Urinary NGAL was purified by tangential flow filtration and ion exchange chromatography. Isolated NGAL was analyzed by SDS-PAGE, immunoblotting and mass spectrometry (MS). The relative molecular mass of NGAL is 23674Da. Peptide mass fingerprinting of the purified NGAL yielded peptides that partially matched with known sequence of P80188 (NGAL_HUMAN). The tryptic digestion profile of isolated NGAL infers that it may be unique and additive molecule in the dictionary of urinary proteins. This is the first report of purification and validation of urinary NGAL from large volume sample by using tangential flow filtration and peptide sequencing respectively. This cost-effective and simplified approach to purification of NGAL, together with the easy availability of urine sample makes the large-scale production of NGAL possible, allowing exploration of various bioclinical as well as biodiagnostic applications.

KEYWORDS:

Acute kidney injury; Immunoblotting; Ion exchange chromatography; NGAL; Tangential flow filtration

PMID:
28324833
DOI:
10.1016/j.jchromb.2017.03.010
[Indexed for MEDLINE]

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