Hrp linked mouse anti human igg

HT-29 cells were stimulated with the different concentrations of κ-carrageenan for 24 h or were pretreated with the indicated concentrations of κ-carrageenan for 1 h and then added to LPS (1 μg/mL) for 24 h. Nuclear extracts were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce, Rockford, IL, USA) according to manufacturer's protocol. Protein concentrations were determined by the Bio-Rad DC Protein assay reagent.
Proteins (30 μg) from nuclear extracts and whole cell lysates were separated by 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis and were measured with antibodies of against TLR4, Bcl10, NF-κB (CST, Danvers, MA, USA), or phospho-IκBα (Abcam, Cambridge, UK), evaluated using the relevant HRP-linked mouse anti-human IgG (Abcam, Cambridge, UK). The immunoreactive proteins were visualized by enhanced chemiluminescence (ECL) kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The results were quantified using NIH Image program by measuring the band intensity and compared to corresponding band intensity of β-actin.

Polystyrene 96-well plates were coated with recombinant proteins (SP17, AKAP4 or PTTG1, from Novus Biologicals, LLC, Littleton, CO, USA) in 50 μL carbonate coating buffer (10 μg/well) for 2 hours at 25 °C. After washing twice with 100 μL PBS, plates were blocked with 1% W/V BSA in PBS for 1 hour. BSA was then removed and 50 μL sera (diluted 1:5 in PBS) were added to plates. After 1 hour incubation at 34 °C, plates were washed twice with 100 μL washing buffer (0.05% V/V Tween-20 in PBS), and incubated with HRP-linked mouse anti-human IgG (Abcam, diluted 1:4,000 in PBS). After 1-hr incubation in the dark plates were washed twice with 100 μL washing buffer and then incubated with HRP substrate solution (TMB) in the dark for 5 minutes at 22 °C. Absorbance was read at 450 nm after incubation with 1N HCl. Absorbance of negative controls (antigen-free wells incubated with commercial antibodies) was subtracted to that obtained in wells incubated with sera.