Sephacryl s 200 hr gel filtration column

To produce recombinant proteins, all constructs were transformed into Escherichia coli BL21 (DE3) cells and cultured at 24°C until the optical density at 600 nm reached 0.4. Then, isopropyl β-D-1-thiogalactopyranoside was added to a final concentration of 0.2 mM and cells were further incubated overnight. After cell lysis, bacterial cell extracts were purified using appropriate resins according to the manufacturer’s instruction. For in vitro sumoylation assays, Arabidopsis SAE1, SAE2, SCE1, and AtSUMO1 (with Gly-Gly at the C-terminus) proteins were purified using Ni²⁺-NTA resin (Qiagen). For in vitro pull-down assays, MBP- and GST-tagged proteins were purified using an amylose resin (New England Biolabs) and glutathione-Sepharose 4B (GE Healthcare), respectively. To generate a specific antibody against XopD_Xcc8004, the His-SUMO-XopD_Xcc8004 protein was purified using a Ni²⁺-NTA resin and cleaved with Ubl-specific protease 1 (Ulp1) to remove the His-SUMO tag. After cleavage, proteins were purified by using a Sephacryl S-200 HR gel filtration column (GE Healthcare) to obtain the XopD_Xcc8004 protein alone. Finally, a rabbit polyclonal antibody raised against XopD_Xcc8004 was obtained by affinity purification using a polyvinylidene difluoride membrane as a coupling matrix [32 (link)].

Codon-optimized DNA fragments encoding SAP11 effectors without the signal peptide were subcloned into the SUMO-pET vector and introduced into Escherichia coli BL21 (DE3). N-terminal His-SUMO-tagged SAP11 proteins were expressed and purified by Ni²⁺-NTA resin (Qiagen) according to the manufacturer’s instructions. Then, the proteins were cleaved with ubiquitin-like-specific protease 1 to remove the His-SUMO tag. Recombinant SAP11 effectors obtained using a Sephacryl S-200 HR gel filtration column (GE Healthcare) were prepared for polyclonal antibody production in rabbits. For western blotting, SAP11_AYWB was detected using anti-SAP11_AYWB serum at 1:10000 dilution, SAP11_CaPM was detected using anti-SAP11_CaPM serum at 1:2500 dilution, and SAP11_PnWB and SAP11_OYM were detected using anti-SAP11_PnWB serum at 1:10000 dilution. Amersham ECL reagents were used. Chemiluminescence signals were captured with an ImageQuant LAS 4000 mini imager (GE Healthcare).

The water-soluble astaxanthin binding protein fractions were obtained by gel filtration column chromatography as previously described. Briefly, stressed cells were harvested, and 1.0 g of stressed wet cells were suspended in 9.0 mL of 50 mM Tris-HCl buffer at pH 7.5. Cells were broken by a multi-beads shocker (Yasui Kikai, Osaka, Japan), dissolved in Tris-buffer pH 7.5. Cell extracts (CFEs) were ultracentrifuged at 100,000× g for 2 h to remove cell debris and lipids. The CFEs were passed through a Sephacryl S-200 HR gel filtration column at a flow rate of 1.0 mL/min (1.6 × 60 cm, GE Healthcare, Chicago, IL, USA). The elution profiles were monitored using a photodiode array detector LaChrome Elite software (Hitachi Ltd., Tokyo, Japan), and fractions of orange eluates were collected. Proteins were concentrated by using Amicon Ultra (Merck, Darmstadt, Germany).