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Phospho-S6RP (Ser235/236) Cellular Assay Kit   HTRF® cellular assay kit for measuring phosphorylated S6 ribosomal protein directly in cells Based on our homogeneous and robust HTRF technology, the Phospho-S6RP assay kit is designed for detecting and studying activated S6RP when phosphorylated at Ser235 and 236 directly in whole cells. Using a streamlined mix-and-read no-wash protocol, this kit can be used from basic research to High Throughput drug screening.   Features Simple assay protocols: an alternative to Western Blot or ELISA Homogeneous, direct detection Sensitive, robust and reliable Suitable for exploring endogeneous and over-expressed receptors Can be used in many types of cells, including primary Compatible with many cell types, including primary cells All inclusive: all reagents in one kit   Applications Screening of receptor activation modulators Screening of compounds acting on upstream events Oncology Diabetes   Assay Principle   The Phospho-S6RP assay kit can be run with cell lysates or whole cells.  Upon activation, S6 Ribosomal Protein (S6RP) is phosphorylated on Ser235 and 236, and after the lysis of the cell membrane, phospho-S6RP can be detected using the kit reagents. The assay is based on a sandwich immunoassay, involving two monoclonal antibodies: an anti-phospho-S6RP antibody labelled with Eu3+-cryptate and an anti-S6RP antibody labelled with d2. These antibodies may be pre-mixed and added in a single dispensing step to further streamline the protocol.      The assays can be run under a two-plate protocol, or be further streamlined to a one-step assay.   Two-plate assay protocol Cells are plated, stimulated and next lysed in the same 96-well culture plate. Lysates are then transferred to the assay plate for the detection of phosphorylated S6RP by HTRF reagents. This protocol enables the cells' viability and confluence to be monitored.     One-plate assay protocol Detection of phosphorylated S6RP with HTRF reagents is performed in a single plate used for plating, stimulation and lysis. No washing steps are required. This protocol, designed for HTS, enables miniaturization while maintaining a high quality output.       Simplified pathway  Ribosomes catalyze protein synthesis, and are composed of a small 40S subunit and a large 60S subunit. The cytoplasmic S6 Ribosomal Protein (S6RP) is a component of the 40S subunit. RPS6 is the major substrate of protein kinases in the ribosome. Phosphorylation is induced by a wide range of stimuli, including growth factors, tumor-promoting agents, and mitogens. Dephosphorylation occurs at growth arrest. The protein may contribute to the control of cell growth and proliferation through the selective translation of particular classes of mRNA.       Product Performance   1. Western Blot versus HTRF assay     HeLa cells were grown in a T175 flask at 37°C, 5% CO2 for 1 day. After removal of cell culture medium, 3mL of supplemented lysis buffer were added and incubated for 30 min. Soluble supernatants were collected after 10 min centrifuging. Equal amounts of lysates were used for a side by side comparison of WB and HTRF.HTRF phospho-RPS6 assay shows better sensitivity than the Western Blot method.     2. Rapamycin dose-response on murine NIH-3T3 cells Murine NIH-3T3 cells were seeded in a cell culture-treated 96-well plate at various densities (from 12,500 to 100,000 cells/well). After an overnight serum starvation, increasing concentrations of rapamycin were applied from 2H, followed by a 20% serum stimulation for 30min. After a 30 minute lysis incubation time with 200µL of supplemented lysis buffer, phosphorylated S6RP was measured using the two-plate Phospho-S6RP assay kit protocol. Note: depending on the cell lines used, lysis volume must be optimized (usually from 50µL to 200µL).     3. Rapamycin dose-response on human HEK293 cells Human HEK293 cells were seeded in a cell culture-treated 96-well plate at various densities (from 12,500 to 100,000 cells/well).  After an overnight serum starvation, increasing concentrations of rapamycin were applied from 2H, followed by a 20% serum stimulation for 30min. After a 30 minute lysis incubation time with 200µL of supplemented lysis buffer, phosphorylated S6RP was measured using the two-plate Phospho-S6RP assay kit protocol. Note: depending on the cell lines used, lysis volume must be optimized (usually from 50µL to 200µL).      

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