Amylose-resin pull-down assay

The amylose-resin pull-down assay was performed to check the binding of nucleosomes and kinetochore components to MBP-tagged CENP-C variants and MBP-tagged CENP-W/Halo–CENP-T variants. The proteins were diluted with binding buffer [10 mM Hepes (pH 7.5), 300 mM NaCl, 2.5% glycerol, and 1 mM TCEP] to 3 μM concentration in a total volume of 50 μl and mixed with 20 μl of amylose beads (New England Biolabs). After mixing the proteins and the beads, 20 μl were taken as input. The rest of the solution was incubated at 4°C for 1 hour on a thermomixer (Eppendorf) set to 1000 rpm. To separate the proteins bound to the amylose beads from the unbound proteins, the samples were centrifuged at 800g for 3 min at 4°C. The supernatant was removed, and the beads were washed four times with 500 μl of binding buffer. After the last washing step, 20 μl of 2× SDS-PAGE sample loading buffer was added to the dry beads. The samples were boiled for 5 min at 95°C and analyzed by SDS-PAGE and CBB (Coumassie Brilliant Blue) staining. In assays performed to investigate the interactions of CENP-T to CCAN, the buffer contained 150 mM NaCl, 15 mM Hepes (pH 7.5), 1 mM TCEP, and 0.01% Tween 20. The samples were incubated for 15 min at 1400 rpm and 4°C.