染色质
乙酰化
组蛋白脱乙酰基酶
细胞生物学
组蛋白
化学
染色质免疫沉淀
分子生物学
生物
DNA
生物化学
基因表达
发起人
基因
作者
Arif Ali Chishti,Zhenhai Li,Baohua Liu,Weiguo Zhu
出处
期刊:Methods in molecular biology
日期:2022-10-19
卷期号:: 303-316
标识
DOI:10.1007/978-1-0716-2788-4_20
摘要
The class III histone deacetylase (HDACs) also known as sirtuins (SIRTs 1-7) are ubiquitously expressed, but SIRT7 mainly resides as nucleolar protein. In this chapter a couple of methods are described that are used to detect modulation of SIRT7 in response to DNA damage. SIRT7 is localized in the nucleoli and binds to the chromatin after DNA damage. Therefore, a protocol was optimized by our lab for chromatin fractionation. By this method, the movement of SIRT7 can be detected from the soluble part (cytosol+nucleoplasm) to the solid part (chromatin) of the cell. Change of SIRT7 expression levels, in different cells or after different treatment, can be detected by isolating whole-cell lysate followed by Western blotting. For analyzing binding of SIRT7 to other substrates, we have also optimized manual immunoprecipitation assays by using 1% NP40 buffer. This protocol is very helpful to pull down SIRT7 and associated proteins by using a single buffer. SIRT7 is a deacetylase, and its deacetylation activity can be checked both inside the cell by in vivo deacetylation assay and outside the cell by in vitro deacetylation assays. Recently it was also discovered that SIRT7 has desuccinylase activity which can be detected by histone desuccinylation assay. This chapter provides the methodology of SIRT7 detection in the whole cell lysate, binding of SIRT7 to the chromatin and other proteins for performing deacetylation and desuccinylation activity.
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