Using the PhosphoTracer AKT 1/2/3 (pS473) assay, the length of assay incubation time that was required to detect recombinant phospho-AKT was examined. After 15 mins, phospho-AKT concentrations of approximately 100 pg/mL were readily detected, while after 1 hour incubation, the limit of detection was less than 10 pg/mL. Longer incubation times had little effect on overall limit of detection that was observed.
Detection of AKT 1/2/3 (pS473) in Jurkat cells treated with various concentration of wortmannin, using ab119614.
As shown, using the AKT 1/2/3 assay kits or Western blot, a significant increase in AKT 1/2/3 phosphorylation at Ser473 is detected in MCF7 cells treated with insulin for 15 minutes (+), compared with untreated cells (-), whereas no change in total AKT 1/2/3 levels was observed.