Fluorescent Labeling of Histone Proteins

To generate Alexa Fluor 488 labeled histone H2B (H2B-Alexa488), histone H2B with T112C mutation was dissolved in reaction buffer (25 mM HEPES, pH 7.5, 6 M guanidine, 5 mM TCEP) to a final concentration of 0.5 mM. Then five molar equivalents of Alexa-488-C₅-maleimide (Invitrogen) was added to the reaction mixture. Keep the reaction for 4 h at 37°C. The fluorophore labeled histone was purified by HPLC using preparative C4 column (25 mm × 250 mm, 10 μm, Grace). The purity and identity of the product was confirmed by LC–MS. Deconvolution result was obtained by UniDec software (25 (link)) (Supplementary Figure S4). Same method was applied to generate Cy5 (Cy5 Maleimide Mono-reactive Dye, GE) labeled histone H2A at K119 position (Supplementary Figure S5).