Fluorescent Labeling of H2A-H2B Histone

This assay follows the labelling methodology previously described in22 (link). The reaction was conducted in the presence of H2A-H2B/T122C (104 μM) with Alexa Flour 488 C5 Maleimide (558 μM (1 mg) dissolved in 200 µl DMSO) (Thermo Fisher), at room temperature for 2 h, in a reaction solution containing 10 mM Tris-HCl pH 7.5, 2 M NaCl, 1 mM EDTA, 1 mM TCEP. The reaction was terminated by the addition of 159 mM 2-mercaptoethanol, followed by dialysis against 10 mM Tris-HCl pH 7.5 buffer containing 2 M NaCl, 1 mM EDTA, and 5 mM 2-mercaptoethanol.

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Matsumoto S., Cavadini S., Bunker R.D., Grand R.S., Potenza A., Rabl J., Yamamoto J., Schenk A.D., Schübeler D., Iwai S., Sugasawa K., Kurumizaka H, & Thomä N.H. (2019). DNA damage detection in nucleosomes involves DNA register shifting. Nature, 571(7763), 79-84.

Publication 2019

Alexa Flour 488 C5 Maleimide DMSO)

Addition Assay Dialysis Dmso Edta Flour Maleimide Mercaptoethanol Mm 159 Nacl Tcep Tris buffer

Corresponding Organization :

Other organizations : Friedrich Miescher Institute, Osaka University, Kobe University, The University of Tokyo, University of Basel

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Variable analysis

independent variables

H2A-H2B/T122C (104 μM)
Alexa Flour 488 C5 Maleimide (558 μM (1 mg) dissolved in 200 µl DMSO)

dependent variables

Labeling of H2A-H2B/T122C

control variables

Reaction solution containing 10 mM Tris-HCl pH 7.5, 2 M NaCl, 1 mM EDTA, 1 mM TCEP
Dialysis against 10 mM Tris-HCl pH 7.5 buffer containing 2 M NaCl, 1 mM EDTA, and 5 mM 2-mercaptoethanol

controls

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

Annotations

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