TY - JOUR
T1 - In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays
AU - Bobde, Ruchir C.
AU - Saharan, Ketul
AU - Baral, Somanath
AU - Gandhi, Surajit
AU - Samal, Archana
AU - Sundaram, Rajivgandhi
AU - Kumar, Ashish
AU - Singh, Ajit K.
AU - Datta, Aritreyee
AU - Vasudevan, Dileep
N1 - Publisher Copyright:
© 2021 JoVE Journal of Visualized Experiments.
PY - 2021/12/29
Y1 - 2021/12/29
N2 - Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting of two copies of the core histones H2A, H2B, H3, and H4, wrapped around by the DNA. The octamer is composed of two copies of an H2A/H2B dimer and a single copy of an H3/H4 tetramer. The highly charged core histones are prone to non-specific interactions with several proteins in the cellular cytoplasm and the nucleus. Histone chaperones form a diverse class of proteins that shuttle histones from the cytoplasm into the nucleus and aid their deposition onto the DNA, thus assisting the nucleosome assembly process. Some histone chaperones are specific for either H2A/H2B or H3/H4, and some function as chaperones for both. This protocol describes how in vitro laboratory techniques such as pull-down assays, analytical size-exclusion chromatography, analytical ultra-centrifugation, and histone chaperoning assay could be used in tandem to confirm whether a given protein is functional as a histone chaperone.
AB - Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting of two copies of the core histones H2A, H2B, H3, and H4, wrapped around by the DNA. The octamer is composed of two copies of an H2A/H2B dimer and a single copy of an H3/H4 tetramer. The highly charged core histones are prone to non-specific interactions with several proteins in the cellular cytoplasm and the nucleus. Histone chaperones form a diverse class of proteins that shuttle histones from the cytoplasm into the nucleus and aid their deposition onto the DNA, thus assisting the nucleosome assembly process. Some histone chaperones are specific for either H2A/H2B or H3/H4, and some function as chaperones for both. This protocol describes how in vitro laboratory techniques such as pull-down assays, analytical size-exclusion chromatography, analytical ultra-centrifugation, and histone chaperoning assay could be used in tandem to confirm whether a given protein is functional as a histone chaperone.
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U2 - 10.3791/63218
DO - 10.3791/63218
M3 - Article
C2 - 35037657
AN - SCOPUS:85123654798
SN - 1940-087X
VL - 2021
JO - Journal of Visualized Experiments
JF - Journal of Visualized Experiments
IS - 178
M1 - e63218
ER -