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 PRAS40 plate is coated with a specific mouse monoclonal PRAS40 capture antibody on the microwells of the 96-well clear plate. Sample lysate or the standard included in the kit are incubated in the microwells allowing PRAS40 antigen 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-PRAS40 (Thr246) antibody to detect the captured PRAS40 on the plate well that is phosphorylated on Thr246. The unbound detection antibody is washed away followed by incubation with an HRP-conjugated anti-rabbit antibody. After the addition of TMB substrate and stop solution the absorbance is measured at 450 nm using a plate reader. This allows for a sensitive enzymatic detection of the sample.
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 generate a standard curve for phosphorylated PRAS40 (Thr246) measurement.