The UPSTATE® colorimetric STAR (Signal Transduction Assay Reaction) ELISA kit is a solid phase sandwich enzyme linked immunosorbent assay that provides a fast, sensitive method to detect specific levels of signaling targets in whole cell extracts. The IR plate is coated with a specific mouse monoclonal IR (β subunit) capture antibody on the microwells of the 96-well clear plate. Sample lysate or the standards included in the kit are incubated in the microwells allowing IR to be captured in the plate wells. The plate is then washed to remove any unbound non-specific material. The wells are then incubated with a specific rabbit anti-phospho-IR antibody to detect the captured IR phosphorylated on Tyrosines 1162 and 1163. The unbound detection antibody is washed away followed by incubation with an HRP-conjugated anti-rabbit antibody. This allows for a sensitive enzymatic detection of the sample. After the addition of TMB substrate and stop solution the absorbance is measured at 450 nm using a plate reader.
The entire assay takes less than 5 hours to complete with minimal hands-on time. Many of the reagents are supplied in ready-to use formulations for ease of use. The kit also includes a standard that is run as both a positive control and to develop a standard curve.