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

• Urinary siderocalin called neutrophil gelatinase-associated lipocalin (NGAL) was purified using a new protocol.
• Detection of urinary NGAL was achieved by immunoblotting using NGAL specific monoclonal antibody.
• Peptide mass fingerprint analysis of NGAL was fairly matched with known sequence of P80188.
• This is the first report of purification of urinary NGAL from large volume urine sample.

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 23674 Da. 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.