Toyopearl superq 650m

Anion-exchange chromatography (Toyopearl SuperQ‐650M; Tosoh Bioscience, Tokyo, Japan) was used for separation. Gel filtration (Sephadex G‐10; GE Healthcare) was used for desalting. Protocols are provided in the supplemental material. Concentration was performed with a rotary evaporator (Laborota 4000; Heidolph, Schwabach, Germany) at 40°C at 2 × 10³ Pa. Freeze-drying was done with a Christ Alpha 1-4 lyophilizer (B. Braun Biotech International, Melsungen, Germany). To prevent degradation of the sugar nucleotides, the sample pH was adjusted to 4.

Ultra-grade HEPES (N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)) and H₃BO₃ were from Sigma-Aldrich Co. (St. Louis, CA, USA) and Fluka (Munich, Germany), respectively. Biochemical grade Tris (tris(hydroxymethyl)aminomethane) and ultra-grade TCA (trichloroacetic acid) were purchased from Panreac AppliChem (Darmstadt, Germany). Calcium chloride standard solution was from Fluka (Munich, Germany). Strontium chloride standard solution, ultra-grade KOH and EDTA (ethylenediaminetetraacetic acid) acid were from Sigma–Aldrich Co. (St. Louis, CA, USA). Standard solutions of EDTA potassium salt was prepared as described in [20 (link)]. TOYOPEARL SuperQ-650M was purchased from Tosoh Bioscience (Griesheim, Germany). HiPrep 26/60 Sephacryl S-100 HR was from GE Healthcare (Chicago, IL, USA). Sephadex G-25 was a product of Pharmacia LKB (Uppsala, Sweden). A molecular mass marker for SDS-PAGE was a product of Thermo Scientific (Waltham, MA, USA). d-10-camphorsulfonic acid was from JASCO, Inc. (Tokyo, Japan). Other chemicals were reagent grade or higher. All buffers and other solutions were prepared using ultrapure water (Millipore Simplicity 185 system). Plastics or quartz ware were used instead of glassware, to avoid contamination of protein samples with Ca²⁺. SnakeSkin dialysis tubing (3.5 kDa MWCO) from Thermo Scientific (Waltham, MA, USA) was used.