Additional information for Arthritis Research & Therapy DEK manuscript

Supplemental information:

Buffer B (6M urea, 0.1M NaH2PO4, 10 mM Tris-l, 0.1 M KCl), used for cell lysis and protein elution, was prepared with the addition of imidazole (final concentration 25 mM) and urea (final concentration ~8M).  Qiagen Ni-NTA Superflow resin was equilibrated with modified Buffer B prior to combination with the cell lysate.  The cell lysate-resin combination was incubated at 4oC for 1.5 hr. Purified protein was eluted from the column with Buffer B containing 100-250 mM imidazole.  Presence and size of rDEK bands was verified by Western blotting.  Protein in positive elution fractions was refolded to native structure by serial dialysis against Buffer D (20 mM HEPES, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 20% glycerol) containing stepwise decreasing concentrations of urea (6M x 2 hr, 4M x 2 hr, 2M x 2 hr, no urea x 12 hr).