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    Simplified detection of polyhistidine-tagged proteins in gels and membranes using a UV-excitable dye and a multiple chelator head pair

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    Type
    Article
    Authors
    Raducanu, Vlad-Stefan cc
    Isaioglou, Ioannis cc
    Raducanu, Daniela-Violeta
    Merzaban, Jasmeen cc
    Hamdan, Samir cc
    KAUST Department
    Biological and Environmental Sciences and Engineering (BESE) Division
    Bioscience Program
    Laboratory of DNA Replication and Recombination
    KAUST Grant Number
    CRG6 (URF/1/3432-01-01)
    Date
    2020-07-09
    Online Publication Date
    2020-07-09
    Print Publication Date
    2020-08-21
    Embargo End Date
    2021-07-09
    Submitted Date
    2020-04-30
    Permanent link to this record
    http://hdl.handle.net/10754/664131
    
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    Abstract
    The polyhistidine tag (His-tag) is one of the most popular protein tags used in the life sciences. Traditionally, the detection of His-tagged proteins relies on immunoblotting with anti-His antibodies. This approach is laborious for certain applications such as protein purification, where time and simplicity are critical. The His-tag can also be directly detected by metal ion–loaded N-nitrilotriacetic acid–based chelator heads conjugated to fluorophores, which is a convenient alternative method to immunoblotting. Typically, such chelator heads are conjugated to either green or red fluorophores, the detection of which requires specialized excitation sources and detection systems. Here, we demonstrate that post-run staining is ideal for His-tag detection by metal ion–loaded and fluorescently labeled chelator heads in PAGE and blot membranes. Additionally, by comparing the performances of different chelator heads, we show how differences in microscopic affinity constants translate to macroscopic differences in the detection limits in environments with limited diffusion, such as PAGE. On the basis of these results, we devise a simple approach, called UVHis-PAGE, that uses metal ion–loaded and fluorescently labeled chelator heads to detect His-tagged proteins in PAGE and blot membranes. Our method uses a UV transilluminator as an excitation source, and the results can be visually inspected by the naked eye.
    Citation
    Raducanu, V.-S., Isaioglou, I., Raducanu, D.-V., Merzaban, J. S., & Hamdan, S. M. (2020). Simplified detection of polyhistidine-tagged proteins in gels and membranes using a UV-excitable dye and a multiple chelator head pair. Journal of Biological Chemistry, jbc.RA120.014132. doi:10.1074/jbc.ra120.014132
    Sponsors
    This work was supported by King Abdullah University of Science and Technology through core funding and the Competitive Research Award [Grant CRG6 (URF/1/3432-01-01) to S.M.H.].
    The authors would like to thank Dr. Mohamed A. Sobhy for his critical evaluation of the manuscript and his valuable feedback, as well as Ms. Afnan Shirbini for her help with the artwork in Fig. 1. We are grateful to Prof. Stefan T. Arold (KAUST) for providing access to the time-resolved fluorescence spectrofluorometer. The authors would also like to thank Mr. Salim Sioud and Mr. Najeh Kharbatia from The Analytical Chemistry Core Lab (ACL) of KAUST for their valuable training in using the chemical preparative and analysis instruments.
    Publisher
    American Society for Biochemistry & Molecular Biology (ASBMB)
    Journal
    Journal of Biological Chemistry
    DOI
    10.1074/jbc.ra120.014132
    Additional Links
    http://www.jbc.org/lookup/doi/10.1074/jbc.RA120.014132
    ae974a485f413a2113503eed53cd6c53
    10.1074/jbc.ra120.014132
    Scopus Count
    Collections
    Articles; Biological and Environmental Science and Engineering (BESE) Division; Bioscience Program

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