Sephadex g 25 column

B. thuringiensis strain (pCG6), producing Cry11Aa toxin [45 (link)], was grown in nutrient broth sporulation medium containing erythromycin (25 mg/mL) at 30 °C for 4–5 days up to complete sporulation [46 (link)]. The spores and crystal inclusions were harvested and purified according to the procedure described by previously reported studies [47 (link),48 (link),49 (link)]. The purified Cry11Aa inclusions were activated by trypsin (1:20, w/w) at 37 °C [50 (link)]. The activated Cry11Aa was biotinylated and purified using a Sephadex G25 column according to the manufacturer’s instruction (GE Healthcare Life Science, Pittsburgh, PA, USA).

Vi, freeze dried as the sodium salt, was solubilized in water and H₂O₂ was added to give a final concentration of 2.5 mg/mL Vi and 5% (wt/v) H₂O₂ in water. The mixture was heated at 80±0.5°C for 2h. The mixture was then injected into a Hiscreen Capto Q [GE Healthcare] column (4.7 mL of resin loading up to 100 mg of fragmented Vi mixture) equilibrated with buffer A and populations of different average size were separated using a gradient step method. NaH₂PO₄ 20 mM pH 7.2 and NaH₂PO₄ 20 mM NaCl 1M pH 7.2 were used as buffer A and B respectively. Pools at average size Vi of 8.6 and 43 kDa were eluted at 25 and 37% of buffer B respectively. Each pool was desalted on a Sephadex G-25 column [GE Healthcare] equilibrated with water. The average size of the fragmented Vi pools was determined by HPLC-SEC equipped with a TSK gel 3000 PW_XL column and a TSK gel PW_XL guard column (Tosoh Bioscience). Dextrans (5, 25, 50, 80, 150 kDa) were used as standards (Sigma Aldrich). The mobile phase was 0.1 M NaCl, 0.1 M NaH₂PO₄, 5% CH₃CN, pH 7.2, at the flow rate of 0.5 mL/min (isocratic method for 30 min). HPAEC-PAD was used to measure Vi content [10 (link), 21 (link)]. ¹H NMR was used to verify Vi identity and confirm O-acetylation levels were >60% [10 (link), 21 (link)].

The manufacture of the lectin microarray and data acquisition were performed as described previously [26 (link)–28 (link)]. The proteins isolated from cells or tissue were labeled with Cy3 fluorescent dye (GE Healthcare, Biosciences, Piscataway, NJ, USA) and purified using a Sephadex-G25 column (GE Healthcare). Subsequently, 4 μg of labeled protein was applied to the lectin microarrays and incubated in the chamber at 37 °C for 3 h. After washing and centrifugation, the slides were scanned using a confocal scanner (4000B, AXON Instruments, USA). The fluorescence intensities were extracted by GenePix 7.0 software (Axon). After filtration and normalization, the parallel datasets were compared with each other based on fold changes according to the following criteria: fold changes ≥ 1.50 or ≤ 0.67 and p < 0.05 indicated upregulation or downregulation, respectively. Significant differences in lectin between samples were evaluated using Student’s t test.