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.

   Check our lysis buffer compatibility      Check our species compatibility

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.

What to expect at the bench

This video covers the principles of phospho-HTRF assays and gives guidelines for performing them, using our phospho-ERK assay as an example.

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).

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
S6RP phospho-S235/236 kit - 500 tests64RP6PEG
-
S6RP phospho-S235/236 kit - 10,000 tests64RP6PEH
-
S6RP phospho-S235/236 kit - 50,000 tests64RP6PEY
-

Companion products

DescriptionCat. noProduct insertMSDS
Phospho-total protein blocking reagent - 2 ml64KB1AAC
Phospho-total protein blocking reagent - 6 ml64KB1AAD
Phospho-total protein lysis buffer #1 - 130 mL64KL1FDF
S6RP phospho-S235/236 kit control lysate64RP6TDA
-
-

ドキュメント