Kta purifier 100

Plasmids were extracted from at least 10⁷ yeast cells per sample using Zymoprep Yeast Plasmid Miniprep II (Zymo Research, Irvine, CA) according to the manufacturer’s instructions. E. coli were transformed with yeast plasmids and single colonies were sequenced with primer 5’-GAG GCT CTG CAC AAC CAC TAC ACG. For further assay, scFv-Fc fusion proteins were generated. The yeast display vectors were digested with SfiI enzyme and the scFv cDNA was subcloned into a pFUSE expression vector (pfusehg1fc; Invivogen), which contains the Fc domain of human IgG1.
The expression vectors were transfected into Expi293F cells using ExpiFectamine 293 reagent, and the supernatant was collected 5 days after transfection. A HiTrap Protein A HP column (no. 17-0403-03; GE Healthcare) was used to affinity-capture antibodies using the ÄKTA purifier 100 (GE Healthcare). Antibodies were then eluted with glycine buffer (pH 2.7). Eluted antibody was neutralized with Tris buffer and stored in PBS after buffer exchange using Ultracel 30 kDa (Merck Millipore). The concentration of antibodies was determined by Qubit Protein Assay Kit (Thermo Fisher Scientific).

BALB/c mice were immunized with recombinant SEB produced using BEVS, and hybridomas were generated as described previously [35 (link)]. A panel of hybridomas was screened to select mAbs binding to recombinant SEB via ELISA. Each hybridoma clone secreting a candidate anti-SEB mAb was confluently cultured in a 6-well plate, and total RNA was extracted to synthesize cDNA using random hexamer primers. For PCR amplification of the antibody VH or VL chain, forward and reverse primer sets were synthesized and used as described previously [36 (link)]. Amplicons corresponding to the VH or VL chain were gel-purified and subcloned into pGEM-T Easy Vector (Promega). Colony PCR was performed to determine the correct insertion of the VH or VL gene, and sequencing analysis was conducted by Bioneer. For mAb production, hybridomas were cultured in 100 mL serum-free media (Gibco) for 5 days, and the supernatants were harvested by centrifugation at 4000× g for 30 min and filtered through a 0.22-μm syringe filter. The mAbs were purified using HiTrap^TM MabSelect^TM PrismA column (GE Healthcare) equipped with ÄKTA purifier 100 (GE Healthcare).

Hippocampus tissues were ground into powder in liquid nitrogen using lysis buffer (Roche). Then, the samples were ultrasonically disrupted on ice. Supernatants were collected after centrifugation (10,000g, 30 min, 4°C), and protein concentrations were determined using an enhanced BCA (bicinchoninic acid) Protein Assay Kit (P0010; Beyotime Biotechnology Ltd., Beijing, China), according to the manufacturer's instructions. The protein samples (200 μg) were mixed with dl-dithiothreitol, alkylated with iodoacetamide, and then treated with trypsin (protein-trypsin ratio = 50 : 1, 12 h).
Protein peptides (100 μg) from each group were labeled using an iTRAQ Reagent-8plex Multiplex Kit (AB SCIEX, Framingham, MA, USA). The samples were labeled as 113 (control 1), 114 (control 2), 115 (CUMS 1), 116 (CUMS 2), and 117 (CUMS 3). The labeled samples were pooled and further fractionated offline using the ÄKTApurifier 100 (GE Healthcare Life Sciences) with a strong cation exchange column (PolySULFOETHYL A™; PolyLC Inc., Columbia, MD, USA). The retained peptides were eluted with buffer A (10 mM KH₂PO₄ in 25% ACN (acetonitrile), pH 3.0) and buffer B (10 mM KH₂PO₄ and 500 mM KCl in 25% ACN, pH 3.0) with a flow rate of 0.7 ml/min.