Sb 804 hq

HPLC-SEC was performed with a gel-filtration column (SB-804HQ, Showa Denko, Tokyo, Japan) using an HPLC system, PU-1580i, connected to an MD1515 multiwavelength detector (JASCO), as described previously [17 (link)]. CgH-spB1WTDelN or CgHspB1WTDelC was heated at the specified temperature for 30 min, loaded onto a column heated at the same temperature, and eluted with buffer B with or without 20% ethylene glycol at a flow rate of 1.0 mL/min. The proteins were monitored by the absorbance at 215 nm.

The chain-length distributions of amylopectin were determined by the SEC coupled with a refractive index (RI) detector. Starch samples and DMSO (50 mg: 10 mL) were mixed and heated at 90 °C with shaking at 160 rpm overnight. Thereafter, 1 mL of sample was transferred and mixed with 6 mL of absolute ethanol. Then, the suspension was separated by centrifugation (5000× g, 20 min) to precipitate the starch granules, and the starch sediment was redissolved in the boiling water for 10 min and then cooled to 50 °C. Sodium acetate (40 μL, 50 mM, pH 3.5) and pullulanase (10 μL, Megazyme, Ireland) were added to the starch solution. Then, the starch solution was debranched at 50 °C for 48 h. The reaction was stopped by boiling for 10 min. Then, the solution (20 μL) was passed through a 0.22 μm membrane filter and injected into the SEC-RID system. Three SEC columns (Shodex OHpak SB-G 6B, SB-804 HQ, and 802.5 HQ) (Showa Denko, Tokyo, Japan) controlled at 60 °C. The system was calibrated with Pullulan P-82 (Showa Denko, Tokyo, Japan). The mobile phase was ammonium acetate (50 mM, pH 5.2) and the flow rate was 1.0 mL/min.

Size exclusion chromatography was performed with a gel‐filtration column (SB‐804HQ; Showa Denko, Tokyo, Japan) using an HPLC system, PU‐1580i, connected to a MD1515 multiwavelength detector (JASCO) as described previously 31. CgHspB1WT or CgHspB1S15D was diluted to the specified concentrations (as monomer) in buffer B. A 100‐µL aliquot of diluted CgHspB1WT or CgHspB1S15D was heated at the specified temperature for 30 min and then loaded onto a column heated at the same temperature and eluted with buffer B with or without 20% ethylene glycol at a flow rate of 1.0 mL·min⁻¹. The proteins are monitored by the absorbance at 215 nm. To examine the reversibility of the dissociation, CgHspB1WT or CgHspB1S15D preheated at 45 °C for 30 min was analyzed by gel filtration at room temperature after cooling at 25 °C for 30 min.