The absorbance was measured as described above. In addition, the binding activity of the PIC to LPS was investigated using the lymphoblast K-562 cell line and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay according to the method described by Han (2009), with a slight changes. serotype typhimurium inside a concentration-dependent manner. In addition, the PIC restored the proliferation activity of the lymphoblast K-562 cells when co-incubated with pathogenic LPS. These results confirm that the PIC prepared in this study is a potentially valuable functional food material or diet supplement as an alternative to antibiotics that can protect animals K-Ras(G12C) inhibitor 9 from pathogenic bacteria. assays. Materials and Methods Preparation of porcine immunoglobulin concentrate (PIC) New porcine blood was collected from a local slaughterhouse, and immediately mixed with sodium citrate (1%). Plasma was separated from your porcine blood K-Ras(G12C) inhibitor 9 by centrifugation at 10,000for 20 min at 4C (1736R; LaboGene, Korea). Calcium chloride (0.5%) was added to the plasma, which was stirred for 5 min and then incubated for 1 h at space temperature to promote serum separation. Ammonium sulfate (1.7 M) was slowly added to the serum, stirred overnight at 4C, and then centrifuged at 10,000for 20 min. The precipitate was dissolved in distilled water, and the pH was modified to 9. The perfect solution is was filtered through a 10-kDa cutoff membrane using ultrafiltration (Vivaflow 50 System; Sartorius, Germany), and then the pH was modified to 7.4. The final product (PIC) was spray-dried using a pilot spray dryer (Yoojin, Korea). Bacterial strains and cell tradition conditions The bacterial strains used in this study were from the Korean Collection for Type Cultures (Korea) and the Korean Tradition Center of Microorganisms (Korea). The pathogenic bacteria were grown in mind heart infusion medium, columbia broth, marine broth, trypticase soy broth, or reinforced clostridial medium for 18 h at 37C (Table 1). Prior to the experiments, bacteria were subcultured at least three times. For long-term storage, stock cultures were stored in new broth comprising 20% glycerol at ?80C. The lymphoblast K-562 cell collection was purchased from your Korean Cell Collection Standard bank (Korea). These cells were cultured in Roswell Park Memorial Institute 1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (Hyclone), penicillin (100 U/mL), and streptomycin (100 g/mL) at 37C inside a humidified atmosphere of 5% CO2. Table 1. Pathogenic strains used in this study No.BacteriumMediumGram positive1ATCC 13124RCM2ATCC 51846RCM3ATCC 33099TSB4ATCC 25923TSB5ATCC 12228TSB6KCTC 5650BHI7ATCC 25175BHI8ATCC 33478BHIGram negative9ATCC 15988BHI10ATCC 23834CB11ATCC 9637TSB12ATCC 43896TSB13ATCC 25586RCM14ATCC 49046TSB15ATCC 33563TSB16KCTC 11862TSB17KCTC 40253TSB18ATCC 12022TSB19ATCC 9290TSB20ATCC 27562MB21ATCC 33844MB Open in a separate windowpane ATCC, American Type Tradition Collection; KCTC, Korean Collection for Type Cultures; RCM, reinforced clostridial medium; TSB, trypticase soy broth; BHI, mind heart infusion medium; CB, columbia broth; MB, marine broth. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) The protein composition of the purified PIC was analyzed by SDS-PAGE inside a 12.5% acrylamide gel as explained by Laemmli (1970). The samples, which included porcine IgG (Sigma), porcine plasma, PIC, and bovine serum albumin (BSA, Sigma), were mixed with sample buffer (0.125 M Tris-HCl, 4% SDS, 20% glycerol, and 2% -mercaptoethanol, pH 6.8) and heated for 15 min at 98C. Electrophoresis was carried out at 20 mA (per gel) for 1 h using a K-Ras(G12C) inhibitor 9 Mini-Protean? Tetra System and PowerPacTM HV (Bio-Rad, USA), and the gel was stained with Coomassie? amazing blue G-250 (Bio-Rad) for 2 h. The bands within the gel were analyzed having a Molecular Imager? GelDocTM XR plus Imaging system and Image LabTM software (version 5.1; Bio-Rad). Measurement of immunoglobulins The concentrations of IgG and IgM in the purified PIC were measured by enzyme-linked immunosorbent assay (ELISA). All reagents were purchased from Bethyl Laboratories, Inc. (USA). The sample and reference were diluted to an appropriate concentration with sample diluent (50 mM Tris, 0.14 M NaCl, 1% BSA, 0.05 Tween 20, pH 8.0). Flat-bottom 96-well Rabbit Polyclonal to USP32 plates (Nunc) were coated with affinity-purified antibody (diluted in covering K-Ras(G12C) inhibitor 9 buffer), incubated for 1 h at space temperature, and then washed four instances with washing remedy (50 mM Tris, 0.14 M NaCl, 0.05% Tween 20, pH 8.0). The remaining binding sites were blocked with obstructing remedy (1% BSA in 50 mM Tris, 0.14 M NaCl, pH 8.0) for 30 min, and then the plate was washed four instances. The diluted sample and stand- ard were added to the plate, which was incubated for 1 h, and then washed four instances. An HRP detection antibody was added to each well and incubated for 1 h. The.
The absorbance was measured as described above
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