Horseradish peroxidase hrp conjugated anti mouse igg

The ability of rTsTryp to bind to IECs was determined by ELISA [20 (link)]. IEC cells were digested by trypsin and collected by centrifugation at 300 g for 5 min. IEC soluble proteins were prepared through cell lysis and sonication on ice, the concentration of IECs soluble protein was determined as reported before [42 (link)]. A 96-well plate (Corning, USA) was coated with IEC proteins at different concentrations (0.125, 0.25, 0.5, 0.75, 1, 1.25, 1.5, and 2 μg/ml) overnight at 4°C, and the plate was blocked with 5% skim milk in PBST at 37°C for 1 h. After washing with PBST, the plate was incubated with 2 μg/ml rTsTryp at 37°C for 2 h, and then probed with 1:100 dilutions of anti-rTsTryp serum, anti-TRX tag serum, infection serum, and normal mouse serum at 37°C for 1 h. After washes with PBST again, the plate was incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (1:10 000, Southern Biotech., USA). Finally, the absorbance at 492 nm was measured after color development and termination [12 (link),13 (link)]. After the optimal IEC protein coating concentration (1.5 μg/ml) was ascertained, the plate was coated with 1.5 μg/ml IEC protein, and incubated with different concentrations of rTsTryp (0.25, 0.5. 0.75, 1, 1.25, 1.5, 1.75, and 2 μg/ml). The subsequent procedures were the same as above-mentioned.

Antibodies against ERK, phosphorylated ERK, p38, phosphorylated p38 MAPKs were purchased from Santa Cruz Biotechnology, Inc. Specific antibodies to COX-1 and COX-2 were purchased from Cell Signaling Technology. Anti-β-actin antibody was obtained from Sigma-Aldrich. Secondary antibodies were horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Southern Biotech) and anti-rabbit IgG (Koma Biotech). PGE₂ was purchased from Cayman Chemical, and IL-1β from R&D Systems. All the siRNA duplexes were obtained from Ambion Inc. as described previously [19 (link)]. PD098059 and SB203580 were purchased from A.G. Scientific, Inc.