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A Quick and Colorful Method to Measure Low-Level Contaminations of Paramagnetic Ni2+ in Protein Samples Purified by Immobilized Metal Ion Affinity Chromatography
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Physical Chemistry. Univ Penn, Dept Biochem & Biophys, Perelman Sch Med, Philadelphia, PA 19104 USA.
Univ Penn, Dept Biochem & Biophys, Perelman Sch Med, Philadelphia, PA 19104 USA.
2019 (English)In: BIOLOGICAL NMR PT A / [ed] Wand, A J, ELSEVIER ACADEMIC PRESS INC , 2019, p. 87-106Chapter in book (Refereed)
Abstract [en]

Isotopic labeling of recombinantly expressed proteins is generally required for investigation by modern nuclear magnetic resonance (NMR) methods. Purification strategies of the labeled proteins often include the use of a polyhistidine affinity tag (His-tag) and immobilized metal ion affinity chromatography (IMAC). Described herein are rapid and inexpensive qualitative and quantitative assays to determine the concentration of paramagnetic Ni2+ in protein samples purified by IMAC. Both qualitative and quantitative colorimetric methods detect the amount of Ni2+ via the color change produced when a [Ni(PAR)(n)](2+) (PAR = 4-(2-pyridylazo)resorcinol, n = 1, 2) complex is formed. The qualitative assay provides a rapid visual test for the presence of Ni2+ in the low micromolar range in a sample of interest. The usefulness of the spectroscopic quantitative assay is illustrated by: (i) detecting a 12 mu M Ni2+ contamination in an NMR sample containing 950 mu M of the 7.5kDa alpha W-3 protein purified by a standard His-tag Ni2+/IMAC approach and (ii) showing that the N-15-HSQC spectrum of the alpha W-3 NMR sample, containing 1 paramagnetic Ni2+ ion per 80 protein molecules, displays clear line broadening of both water and protein spectral lines. We also (iii) measured Ni2+ release during the equilibration, wash, and elution steps of three commonly used Ni2+/IMAC resins when following manufacturer's protocols. The concentration of Ni2+ detected in elutes of the three resins ranged from 2 mu M to nearly 1mM.

Place, publisher, year, edition, pages
ELSEVIER ACADEMIC PRESS INC , 2019. p. 87-106
Series
Methods in Enzymology, ISSN 0076-6879 ; 614
National Category
Biophysics
Identifiers
URN: urn:nbn:se:uu:diva-401959DOI: 10.1016/bs.mie.2018.08.037ISI: 000500377800005PubMedID: 30611434ISBN: 978-0-12-813860-1 (print)OAI: oai:DiVA.org:uu-401959DiVA, id: diva2:1384512
Available from: 2020-01-10 Created: 2020-01-10 Last updated: 2020-01-10Bibliographically approved

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