Native gel shift assays

One protein component was held constant at 10 µM and the other was titrated. Samples were separated by 5% polyacrylamide gel electrophoresis. Gels were run for 100 min at 150 V at 4°C in 0.5x TBE (40 mM Tris-HCl pH 8.4, 45 mM boric acid, 1 mM EDTA). Gels were stained with Coomassie Blue.

Nap1, Imp9 or Imp9-Ran (equimolar Imp9 and RanGTP added together without further purification) were titrated (at 0.5, 1.0 and 1.5 molar equivalents of H2A-H2B) against 147 bp Widom 601 DNA mixed with H2A-H2B at 1:7 (1.5 µM:10.5 µM), or 147 bp Widom 601 DNA was titrated against 10.5 μM Nap1, Imp9 or Imp9-Ran (1:1) pre-mixed with an equimolar amount of H2A-H2B. Samples were separated by 5% polyacrylamide gel electrophoresis. Gels were run for 75 min at 150 V at 4°C in 0.5x TBE. Gels were stained with ethidium bromide and then Coomassie Blue.

Tetrasomes containing H3-H4 and 147 bp Widom 601 DNA were reconstituted as described in Dyer et al (Dyer et al., 2004). To monitor nucleosome assembly, tetrasomes were held constant at 2.5 µM and H2A-H2B or pre-formed complexes of Nap1-H2A-H2B (1:1), Imp9-H2A-H2B (1:1), or Imp9-H2A-H2B-Ran (1:1:1) were titrated. To monitor nucleosome disassembly, Nap1, Imp9, or Imp9-Ran (1:1) complex was titrated against nucleosomes (2.5 µM). Samples were separated by 5% polyacrylamide gel electrophoresis. Gels were run for 75 min at 150 V at 4°C in 0.5x TBE. Gels were stained with ethidium bromide and then Coomassie Blue.