Phospho-S6RP (Ser235/236) cellular kit HTRF®

  • Ready-to-use Ready-to-use
  • High sensitivity High sensitivity
  • Faster and more convenient than ELISA Faster and more convenient than ELISA
  • No-wash No-wash


The Phospho-S6RP (Ser235/236) cellular assay kit enables the quantitative detection of phosphorylated S6 ribosomal protein when phosphorylated on Serine 235/236. It can be used for the screening of receptor activation modulators and compounds acting on upstream events. This kit has various applications, from inflammation/immunology to metabolism/diabetes and oncology research.



Phospho-S6RP (Ser235/236) assay principle

The Phospho-S6RP (Ser235/236) assay measures S6RP when phosphorylated at Ser235/236. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-S6RP (Ser235/236) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein’s phosphorylation state under a no-wash assay format.
Phospho-S6RP (Ser235/236) assay principle

Phospho-S6RP (Ser235/236) 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding phospho-S6RP (Ser235/236)HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-S6RP (Ser235/236) 2-plate assay protocol

Phospho-S6RP (Ser235/236) 1-plate assay protocol

Detection of phosphorylated p-S6RP with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.
Phospho-S6RP (Ser235/236) 1-plate assay protocol

HTRF compared to WB using phospho-S6RP (Ser235/236) cellular assay kit

HeLa cells were grown in a T175 flask at 37°C, 5% CO2 for 1 day. After removal of cell culture medium, 3 mL 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-S6RP assay shows better sensitivity than the Western Blot method.
HTRF compared to WB using phospho-S6RP (Ser235/236) cellular assay kit

Rapamycin dose response on murine NIH-3T3 or HEK293 cells

Murine NIH-3T3 or 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 minutes 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).
Rapamycin dose response on murine NIH-3T3 cells

Phospho-S6RP 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. S6RP protein may contribute to the control of cell growth and proliferation through the selective translation of particular classes of mRNA.
Phoshpo-S6RP signaling pathway

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Open R&D: Sanofi Access Platform

In collaboration with Sanofi - Scientific Presentations

Lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

p-S6RP, p-p70S6K and p-EIF4E - Validation of three new HTRF® phospho-assays for the study of translational control.

p-S6RP, p-p70S6K and p-EIF4E - Validation of three new HTRF® phospho-assays for the study of translational control. - Posters

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

HTRF Product Catalog

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection

Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes

Assessment of drug efficacy and toxicity by combining innovative technologies

Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Application Notes

Plate Reader Requirement

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