Resource q anion exchange column

The modified antibody and oligonucleotide were mixed at a ratio of 1:4 by active groups (the determination of the active groups’ content is described below). The reaction mixture was kept at room temperature overnight. The purification was carried out in several stages. To remove the unreacted antibody, the reaction mixture was placed in a RESOURCE Q anion exchange column (GE Healthcare). Chromatography was carried out in 50 mM TrisHCl at pH 7.5 with a linear gradient of NaCl and a flow rate of 1.5 ml/min. The resulting fractions were concentrated and placed in a Superdex 200 10/30 Increase column (GE Healthcare). Chromatography was carried out again in 0.1× PBS buffer at a flow rate of 0.5 ml/min. The fractions were evaporated in a vacuum centrifuge and analyzed by SDS-PAGE in 6% gel (in non-reducing conditions without the addition of mercaptoethanol) and 10% gel (in reducing conditions with the addition of mercaptoethanol), followed by silver staining.

ETEC H10407 supernatants were fractionated using a AKTA Fast Protein Liquid Chromatography (FPLC) system (GE Life Sciences, Marlborough, MA, USA) to facilitate molecular characterization of the ESF. Acetone-precipitated supernatants from M9-grown ETEC cultures were resuspended in 10 mL 0.15 M NaCl, clarified by 0.22 µm filtration, and applied at 4 mL/min to a Superdex S200 26/60 column (GE Life Sciences) that had previously been equilibrated in 20 mM Tris (pH 8.0), 200 mM NaCl. The ESF-containing eluent was collected from the column between 110–130 mL, and dialyzed against 4 L of 20 mM Tris (pH 8.0) in preparation for further chromatography. The ESF-containing sample was applied to a 1 mL Resource Q anion exchange column (GE Life Sciences). The column was washed with 20 mM Tris (pH 8.0) until the OD₂₈₀ value reach baseline, and then the bound proteins were eluted with a gradient to 1 M NaCl in the same buffer. Fractions of 1 mL were collected and then screened for their ability to prevent IκBα degradation in response to TNF, separated by SDS-PAGE, and detected using Silver Staining (Thermo Scientific, Waltham, MA, USA). Proteins from active fractions were excised and digested in-gel with trypsin (Promega, Madison, WI, USA). Proteins were identified using mass spectrometry at the Mass Spectrometry & Analytical Proteomics Laboratory, University of Kansas.

Genes coding for CT region of human Na_V (CTNa_V) isoforms, namely, CTNa_V1.4T (amino acids 1599 to 1764), CTNa_V1.4FL (amino acids 1599 to 1836), CTNa_V1.5T (amino acids 1775 to 1940), CTNa_V1.5FL (amino acids 1775 to 2016), CTNa_V1.7T (amino acids 1761 to 1928), CTNa_V1.7FL (amino acids 1761 to 1988), CTNa_V1.9T (amino acids 1605 to 1768), and CTNa_V1.9FL (amino acids 1605 to 1791), were subcloned into pGEX 6p1 expression vector (GenScript) and coexpressed with mammalian CaM in BL21-CodonPlus RIL E. coli cells (Agilent), purified using GST-sepharose 4b resin (GE Lifesciences), ReSource Q anion exchange column (GE, MilliporeSigma).

Protein was expressed in E. coli. Cells were grown at 37 °C until an OD of 0.8 and then induced with 200 μM IPTG. Temperature was set to 18 °C and protein was expressed overnight. Cells were collected in lysis buffer (50 mM TRIS pH 8, 100 mM NaCl, 1 mM β-mercaptoethanol) with Complete EDTA-free protease inhibitor (Sigma) and lysed by sonication. Lysate was cleared by spinning down at 21,000 × g and loaded on TALON beads (Clontech Laboratories, Inc.). Beads were washed with 20 complete volumes  of lysis buffer +6 mM Imidazole and eluted in lysis buffer + 300 mM Imidazole. Sample was diluted to 50 mM NaCl and injected into a Resource Q anion exchange column (GE Healthcare). Protein was eluted using a linear salt gradient ranging from 50 mM NaCl to 600 mM NaCl. In a final step, the sample was purified on a Superdex 200 size exclusion column (GE Healthcare) in lysis buffer. Samples were concentrated and stored at − 80 °C.

The coding sequence cDNA of ShKAI2iB was amplified by PCR using total complementary DNA from the total RNA of 1-day conditioned Striga seeds. ShKAI2iB (1–270, C270S) was designed for crystallization and other assays. For expression in Escherichia coli, the PCR product was cloned into an expression vector pGEX-6P-3 (GE Healthcare) and subsequently transformed into E. coli strain Rosetta (DE3) (Novagen). IPTG-induced overexpression was performed for 20 h at 25 °C. For purification, cell pellets were lysed by sonication in buffer A (20 mM Tris-HCl, pH 8.0, 0.3 M NaCl, 1 mM DTT), and the soluble fraction separated by centrifugation was purified using Glutathione Sepharose 4B resin (GE Healthcare) and a Resource Q anion-exchange column (GE Healthcare). ShKAI2iB mutants were overexpressed and purified with the same procedure as wild-type protein. For crystallization, buffer exchange accompanied by concentration was performed using Vivaspin 20 (5,000 MWCO PES) (Sartorius). Purified ShKAI2iB was dialyzed against buffer B (20 mM HEPES-NaOH, pH 8.0, 50 mM NaCl) for the ITC experiments and the intrinsic fluorescence assay.