A Quick and Colorful Method to Measure Low-Level Contaminations of Paramagnetic Ni2+ in Protein Samples Purified by Immobilized Metal Ion Affinity Chromatography. Academic Article uri icon

abstract

  • 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 12M Ni2+ contamination in an NMR sample containing 950M of the 7.5kDa 3W protein purified by a standard His-tag Ni2+/IMAC approach and (ii) showing that the 15N-HSQC spectrum of the 3W 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 2M to nearly 1mM.

published proceedings

  • Methods Enzymol

author list (cited authors)

  • Glover, S. D., & Tommos, C.

complete list of authors

  • Glover, Starla D||Tommos, Cecilia

publication date

  • January 2019