Horseradish peroxidase conjugated goat anti rabbit igg

The purified anti-corisin IgG antibody was used at 1/1000 dilution for western blotting in lung tissue. We measured the concentration of corisin in body fluids using a competitive enzyme immune assay. Briefly, the purified corisin from transglycosylase 351 was coated on a 96-well plate at a final concentration of 2 µg/ml in phosphate-buffered saline at 4 °C overnight. After blocking and appropriate washing, the standards, samples and 5 ng/ml of anti-corisin were added to the wells and incubated at 4 °C overnight. The wells were then washed before adding horseradish peroxidase-conjugated goat anti-rabbit IgG (R&D System), as the secondary antibody, in a phosphate-buffered saline solution containing 5 µg/mL human IgG. After appropriate washing and incubation, substrate solution was added for color development and absorbance read at 450 nm. Values were extrapolated from a standard curve prepared using several concentrations of the peptide.

Soluble proteins were extracted from cells, and western blot analysis was performed as described previously [14 (link)]. Specific antibodies for cyclin D1 (1:1000 dilution, Santa Cruz, Inc.), cyclin E (1:2000 dilution, Novus Biologicals, Inc.) and CDK2 (1:2000 dilution, Novus Biologicals, Inc.) were used as primary antibodies. Horseradish peroxidase-conjugated goat anti-rabbit IgG was the secondary antibody (R&D Systems, Cat. BAF008). GAPDH served as an internal control.

Protein was isolated from homogenized heart tissue following standard protocols. Total proteins were loaded onto an SDS-PAGE gel and transferred electrophoretically to nitrocellulose membranes (Millipore, Billerica, MA). After blocking with 5% skim milk, the membranes were incubated with the appropriate primary antibody of the recommended dilution at 4°C overnight. The membranes were washed and further incubated with horseradish peroxidase—linked secondary antibody at 37°C for 60 min. The blots were developed with an enhanced chemiluminescence reagent kit (Millipore, Billerica, MA) and visualized with UVP Bioimaging Systems. Vision Works LS Acquisition and Analysis Software were used to analyze blot densities.
Primary antibodies used in this study were as follows: OSM (R&D systems, Minneapolis, MN), Oβ (R&D systems, Minneapolis, MN), cleaved caspase 3 (Cell Signaling, USA), caspase 3 (Cell Signaling, USA), β-actin (Cell Signaling, USA), p-Akt (Cell Signaling, USA), Akt (Cell Signaling, USA), VEGF (R&D systems, Minneapolis, MN), and bFGF (UBI, Lake Placid, NY). Secondary antibodies were horseradish peroxidase-conjugated goat anti-rabbit IgG (R&D systems, Minneapolis, MN) and rabbit anti-goat IgG (R&D systems, Minneapolis, MN).