Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Dec;30(4):351-359.
doi: 10.1007/s10577-022-09709-1. Epub 2022 Nov 18.

Ellman's reagent prevents dephosphorylation of histones during isolation of mitotic chromosomes

James R Paulson  1 , Erica R Vander Mause  2   3 , Elizabeth Dillinger  2   4 , Megan E Luedeman  2   5 , Bakhtawar Usman  2   6
Affiliations

Ellman's reagent prevents dephosphorylation of histones during isolation of mitotic chromosomes

James R Paulson et al. Chromosome Res. 2022 Dec.

Abstract

Histones H1 and H3 are highly phosphorylated in mitotic HeLa cells but are rapidly dephosphorylated by endogenous protein phosphatases during the isolation of metaphase chromosomes. We show that this dephosphorylation can be prevented by including the sulfhydryl reagent 5,5'-dithiobis-(2-nitrobenzoate) (Ellman's reagent, or DTNB) in the isolation buffer. The minimal amount of DTNB required is approximately stoichiometric with the number of sulfhydryl groups in the lysate. Inhibition of the protein phosphatases can subsequently be reversed by treatment with dithiothreitol or 2-mercaptoethanol. DTNB is compatible with the isolation of either metaphase chromosome clusters or individual metaphase chromosomes. It should be useful in investigations of the structure and biochemistry of chromatin and chromosomes and in the study of possible functions for mitotic histone phosphorylation.

Keywords: Chromosome structure; Histone H1; Mitosis; Phosphatases; Protein phosphorylation.

PubMed Disclaimer

References

    1. Bannister AJ, Kouzarides T (2011) Regulation of chromatin by histone modifications. Cell Res 21:381–395. https://doi.org/10.1038/cr.2011.22 - DOI
    1. Barford D, Das AK, Egloff MP (1998) The structure and mechanism of protein phosphatases: insights into catalysis and regulation. Annu Rev Biophys Biomol Struct 27:133–164. https://doi.org/10.1146/annurev.biophys.27.1.133 (PMID: 9646865) - DOI
    1. Csortos C, Matkó J, Erdödi F, Gergely P (1990) Interaction of the catalytic subunits of protein phosphatase-1 and 2A with inhibitor-1 and 2: a fluorescent study with sulfhydryl-specific pyrene maleimide. Biochem Biophys Res Commun 169:559–564. https://doi.org/10.1016/0006-291x(90)90367-v - DOI
    1. D’Anna JA, Gurley LR, Deaven LL (1978) Dephosphorylation of histones H1 and H3 during the isolation of metaphase chromosomes. Nucleic Acids Res 5:3195–3207. https://doi.org/10.1093/nar/5.9.3195 - DOI
    1. Earnshaw W, Fujimori E (1973) Dark bleaching of rhodopsin by organic mercurial. FEBS (Fed Eur Biochem Soc) Lett 34:137–139. https://doi.org/10.1016/0014-5793(73)80775-6 .

Publication types

LinkOut - more resources