GR-Loading Complex Formation and Characterization

Using reaction buffer containing 50 mM HEPES pH 7.5, 50 mM KCl and 2 mM DTT, 10 μM Hsp90 dimer of ATP-binding-deficient mutant (Hsp90(D93N))^{46 (link)}, 10 μM Hop, 15 μM Hsp70, 4 μM Hsp40 and 20 μM MBP-GRLBD were incubated with 5 mM ATP/MgCl₂ for 1 h at room temperature. The complex was purified and analysed by size-exclusion chromatography with multi-angle light scattering (SEC-MALS) with a Wyatt 050S5 column on an Ettan LC (GE Healthcare) in a running buffer containing 50 mM HEPES, 50 mM KCl, 5 mM MgCl₂, 2 mM DTT, 200 μM ADP and 0.01% octyl β-d-glucopyranoside (β-OG). Molecular weights were determined by multi-angle laser light scattering using an in-line DAWN HELEOS and Optilab rEX differential refractive index detector (Wyatt Technology Corporation). Once eluted, fractions containing the GR-loading complex were immediately cross-linked with 0.02% glutaraldehyde for 20 min at room temperature and quenched with 20 mM Tris pH 7.5. Fractions containing the GR-loading complex were separately snap-frozen in liquid nitrogen, and stored in aliquots at −80 °C.