Purified recombinant Hax-1 protein (1 mg for the western blot, indicated amount for silver staining) was suspended in 100 ml of sample buffer (SB: 10 mM HEPES, pH 7.4, 120 mM potassium acetate, 2.5 mM MgCl 2 , 1.6 mM dithiotreitol), incubated for 15 min in room temperature to establish monomer-dimer equilibrium and then incubated for 20 min in 228C with 0.00125% glutaraldehyde (Sigma). The reaction was stopped by the addition of 50 ml of 3-fold concentrated loading buffer (150 mM Tris pH 6.8, 6 mM EDTA, 6% SDS, 30% glycerol, 1 M urea, 0.003% bromophenol blue). Samples were heated (908C) and separated on the SDS-PAGE. Proteins were detected by silver staining (29) and western blot. The lanes in the silver-stained gel were scanned and the areas under the peaks were quantified using Multi-analyst (Bio-Rad). The results were analyzed by nonlinear regression analysis to determine an approximate dimer dissociation constant, as described in (30) .