Western Blot Analysis of Histone Proteins

Western blot analysis was performed as described previously (16 (link)). Briefly, cells were suspended in RIPA buffer (50 mM Tris–HCl (pH 8.0), 420 mM NaCl, 0.5% sodium deoxycholate, 0.1% Sodium dodecyl sulfate, 1% NP-40) and sonicated. The extract was incubated for 30 min on ice, and then incubated at 95°C for 5 min. The extract was loaded and run on SDS-PAGE gel as standard protocols. For histone proteins, intensity was analyzed by OdysseyR CLx Imagins System(LI-COR). Anti-lamin B1 (12987-1-AP, Proteintech), anti-lamin A/C (3A6-4C11, Diagenode), anti-H2Aub (D27C4), anti-Ring1B (monoclonal antibody kindly gifted from Dr Koseki, RIKEN), anti-Ring1A (#2820, CST) were used.

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Fukuda K., Shimi T., Shimura C., Ono T., Suzuki T., Onoue K., Okayama S., Miura H., Hiratani I., Ikeda K., Okada Y., Dohmae N., Yonemura S., Inoue A., Kimura H, & Shinkai Y. (2023). Epigenetic plasticity safeguards heterochromatin configuration in mammals. Nucleic Acids Research, 51(12), 6190-6207.

Publication 2023

OdysseyR CLx Imagins System Anti-lamin B1 (12987-1-AP,

Anti b1 Buffer Cells Histone Lamin Lamin a c Lamin b1 Monoclonal antibody Nacl Np 40 Proteins Ring1b Ripa Sds page Sodium deoxycholate Sodium dodecyl sulfate Tris Western blot analysis

Corresponding Organization : University of Melbourne

Other organizations : Tokyo Institute of Technology, RIKEN Center for Sustainable Resource Science, RIKEN Center for Biosystems Dynamics Research, The University of Tokyo, Tokushima University, RIKEN Center for Integrative Medical Sciences, Tokyo Metropolitan University

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Variable analysis

independent variables

RIPA buffer composition (50 mM Tris–HCl (pH 8.0), 420 mM NaCl, 0.5% sodium deoxycholate, 0.1% Sodium dodecyl sulfate, 1% NP-40)
Sonication of cell extract
Incubation of extract at 95°C for 5 min

dependent variables

Protein expression levels (histone proteins, lamin B1, lamin A/C, H2Aub, Ring1B, Ring1A)

control variables

SDS-PAGE gel electrophoresis
Odyssey CLx Imaging System (LI-COR) for intensity analysis
Antibodies used (anti-lamin B1, anti-lamin A/C, anti-H2Aub, anti-Ring1B, anti-Ring1A)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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