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Current Protocols · 2025 ·Vol. 5 ·期 5 ·e70155 · IF 2.2

Purification of Recombinant Histones and Mononucleosome Assembly

Yiming Zhao,Jingjun Hong

The nucleosome is the basic unit of chromatin. Each nucleosome is composed of a little less than two turns of DNA wrapped around a set of eight proteins called histones, which are known as a histone octamer . Each histone octamer is composed of two copies each of the histone proteins H2A , H2B , H3 , and H4 . Nucleosomes play an important role in gene expression and regulation. While previous protocols use HPLC (high-performance liquid chromatography) to purify each histone and, after octamer reconstitution, subsequent FPLC (fast protein liquid chromatography) to purify the octamers. Here we present a method to carry out octamer reconstitution and mononucleosome assembly with FPLC only. This basic protocol describes a procedure for histone purification from Escherichia coli (E. coli) , their subsequent reconstitution as octamers, and assembly into mononucleosomes in vitro . Through this protocol, histone octamers and mononucleosomes can be reconstituted on a large scale without the use of HPLC. Overall, this protocol describes an effective new method for the reconstitution of octamers and assembly of mononucleosomes. © 2025 Wiley Periodicals LLC. Basic Protocol : Purification of recombinant histones and mononucleosome assembly

CAS / MDL: 110-18-9 115-39-9 329-98-6 367-93-1 56-81-5 60-00-4 3483-12-3 60-24-2

研究领域: 生物化学 · 分子生物学 · 结构生物学

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Current Protocols · 2025 ·Vol. 5 ·期 7 ·e70179 · IF 2.2

In vitro Nucleosome Assembly Facilitated by the Histone Chaperone SRCAP

Bingyan Yuan,Jingjun Hong

In eukaryotic cells, nucleosomes are octameric structures composed of DNA and histones, and their tandem arrangement ultimately forms chromosomes. Nucleosome assembly is governed in vivo by a series of molecular mechanisms, particularly histone chaperones such as Swr1, which is indispensable for H2A.Z-nucleosome formation. The assessment of nucleosome assembly using micrococcal nuclease (MNase) digestion has been widely accepted. We describe a simple and reproducible protocol for analyzing the chaperone activity of SRCAP (Snf2-related CREBBP activator protein), the histone H2A.Z chaperone. This approach involves purifying a complex of H2A.Z, H2B, and SRCAP via fast protein liquid chromatography (FPLC) only, followed by its assembly with DNA-(H3.1-H4 A15C ) 2 . The formation of nucleosomes is then determined in vitro using electrophoretic mobility shift assay (EMSA) and MNase digestion. This approach can also be adapted to evaluate whether other histone chaperones or regulatory factors promote nucleosome assembly activity. © 2025 Wiley Periodicals LLC. Basic Protocol : In vitro nucleosome assembly facilitated by the histone chaperone SRCAP

研究领域: 分子生物学 · 生物化学 · 细胞生物学 · 表观遗传学

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Purification of Recombinant Histones and Mononucleosome Assembly

In vitro Nucleosome Assembly Facilitated by the Histone Chaperone SRCAP