Human/Mouse/Rat Phospho-RPS6 (S235/S236) and Total RPS6 PharmaGenie ELISA Kit (SBRS1954)

Building on the dual-analyte workflow, each Assay Genie kit includes a pre-coated microplate of twelve 8-well strips with anti-pan RPS6 capture antibody, and the 96-assay format divides wells between phospho-S235/S236 detection and total pan-RPS6 detection. The standard protocol calls for a 100 µL sample load per well, a 2.5 hour room-temperature incubation (or overnight step at 4 °C), followed by 1 hour with primary antibody, 1 hour with HRP-conjugated streptavidin, 30 minutes of TMB One-Step Substrate development, 50 µL of Stop Solution, and immediate absorbance readout. Capture and detection antibodies were raised against the conserved RLSSLR C-terminal phospho-epitope of human RPS6 (UniProt P62753, Entrez Gene ID 6194, ~28.7 kDa), and the antibody pair shows cross-reactivity with mouse and rat orthologs in cell and tissue lysates. The kit is applied in PI3K-AKT-mTOR pathway, translation-initiation, insulin signaling, and autophagy research, including rapamycin and serum-stimulation time-course experiments. Lysates are prepared at ~4 × 10^7 cells/mL in the supplied Cell Lysate Buffer plus protease and phosphatase inhibitors, cleared by microcentrifugation at 13,000 rpm for 10 minutes on ice, aliquoted, and stored at -20°C; avoid repeated freeze-thaw cycles. Reagents ship on blue ice and should be stored at -20°C upon receipt with a shelf life of approximately 6 months; for research use only.