To allow site-specific labelling of histone H2B, a cysteine was introduced via site-directed mutagenesis (H2BK120C, as previously described55 (link)) using mutagenesis primers indicated in Table S1 . H2BK120C was labelled with Cyanine5-maleimide (Lumiprobe cat #13080) as described previously55 (link). Labelled octamers were refolded as described above. Before chromatin reconstitution, the unlabelled and labelled octamers were combined at a molar ratio of 7:1 (unlabeled:labelled). Chromatin was then reconstituted as described above.
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Site-Specific Labeling of Histone H2B
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Corresponding Organization : EMBL Australia
Other organizations : ARC Centre of Excellence in Advanced Molecular Imaging
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Variable analysis
independent variables
- Site-directed mutagenesis to introduce a cysteine at position 120 of histone H2B (H2BK120C)
- Labeling of H2BK120C with Cyanine5-maleimide
- Refolding of labelled octamers
- Reconstitution of chromatin with a molar ratio of 7:1 (unlabeled:labelled) octamers
- Mutagenesis primers indicated in Table S1
- Labeling and refolding procedures described previously (link)
- Chromatin reconstitution method described above
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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