Sephadex g 10 resin

Starch binding was assessed as described previously by Seung et al. (2015) with minor modifications. Recombinant polyhistidine-tagged At-SS5 protein was used at a final concentration of 300 nM and was incubated with 30 mg of hydrated waxy maize starch in 50 mM HEPES-NaOH, pH 7.5, 2 mM MgCl 2 , 1 mM DTT, 0.05% (w/v) BSA, and 0.005% (v/v) Triton X-100 for 45 min at 20°C. Starch was pelleted by centrifugation (30 s, 5000 rcf), and an aliquot of the supernatant was collected as the soluble protein fraction (S). Substrates were then washed and bound proteins were eluted as described by Seung et al. (2015) . Supernatant from the last wash and the elution step were collected as the final wash (FW) and insoluble (I) fractions. S, FW, and I fractions were mixed with SDS-PAGE loading buffer (final concentrations of 100 mM DTT, 3% [v/v] glycerol, 2% [w/v] SDS, 50 mM Tris-HCl, pH 6.8, and 0.005% [w/v] bromophenol blue) and analyzed by immunoblotting (see below). Sephadex G-10 resin (GE Healthcare) was used as a substitute for starch in control samples. The protein portion found in the insoluble fraction was estimated using Fiji (Schindelin et al., 2012) in relation to the respective soluble fraction as previously described by Kesten et al. (2016) .