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. 2017 Jan 5;7(1):e2085.
doi: 10.21769/BioProtoc.2085.

In vitro Histone H3 Cleavage Assay for Yeast and Chicken Liver H3 Protease

Sakshi Chauhan  1 , Gajendra Kumar Azad  1   2 , Raghuvir Singh Tomar  1
Affiliations

In vitro Histone H3 Cleavage Assay for Yeast and Chicken Liver H3 Protease

Sakshi Chauhan et al. Bio Protoc. .

Abstract

Histone proteins are subjected to a wide array of reversible and irreversible post-translational modifications (PTMs) (Bannister and Kouzarides, 2011; Azad and Tomar, 2014). The PTMs on histones are known to regulate chromatin structure and function. Histones are irreversibly modified by proteolytic clipping of their tail domains. The proteolytic clipping of histone tails is continuously attracting interest of researchers in the field of chromatin biology. We can recapitulate H3-clipping by performing in vitro H3 cleavage assay. Here, we are presenting the detailed protocol to perform in vitro H3 cleavage assay.

Keywords: Chicken liver H3 protease; Chromatin; Histone H3; Histone clipping; Yeast H3 protease.

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Figures

Figure 1.
Figure 1.. Purification of core histones
. Core histones were purified from chicken brain tissue through hydroxyapatite resin and the purity was checked by running 18% SDS-PAGE. 2 μg core histones were loaded onto a lane.
Figure 2.
Figure 2.. Western blot of liver microsomal extract with Glud1 antibody
. Size exclusion chromatography fractions of chicken liver microsomal extract probed with Glud1 (Chicken liver H3 protease) antibody to confirm the presence of H3 protease in these fractions.
Figure 3.
Figure 3.. In vitro histone H3 cleavage assay with core histones
. A. In vitro activity assay with yeast protease. B. In vitro activity assay with chicken liver protease. Out of four core histones, only histone H3 get cleaved as protease is specific for histone H3. ‘-’ and ‘+’ indicate absence and presence of H3 protease respectively.
See this image and copyright information in PMC

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References

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