Measuring IMP4 Binding to H3-H4-ASF1 Complexes

K_D values of IMP4 binding to H3–H4–ASF1 complexes were obtained by fluorescence polarization assays, as previously described (32 ). For these assays, the H4_E63C mutant protein (The Histone Source) was labeled with the XFD488 fluorophore (AAT Bioquest) by mixing H4_E63C and the dye at a 1:4 molar ratio in the histone unfolding buffer (50 mM Tris-HCl [pH 7.5] and 5 M guanidine-HCl) without DTT for 4 h at 25 °C in the dark. Labeling efficiency was further confirmed by intact mass spectrometry. H3–H4_E63C tetramers, ASF1–H3_wt–H4_E63C, and ASF1–H3_{31 to 135}–H4_E63C were formed as for the wt complexes. MBP–IMP4 and the preassembled ASF1–H3–H4 complexes were separately dialyzed in buffer containing 50 mM HEPES (pH 7.5), 300 mM NaCl, and 2 mM MgCl₂. MBP–IMP4 was serially diluted from 10 μM to 0.3 nM in the presence of 40 nM ASF1–H3_wt–H4_E63C or ASF1–H3_{31 to 135}–H4_E63C in a 384-well microplate (Corning). Fluorescence signal for each titration was measured, and data were processed in, PALMIST (43 (link)) using averages of triplicate experiments. Final figures were generated with GUSSI (44 (link)). Data were processed in PALMIST (43 (link)) using averages of triplicate experiments, and the final figures were generated with GUSSI (44 (link)).