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HTRF Phospho-RSK1 (T359) Cellular Kit HTRF®

This HTRF kit enables the cell-based quantitative detection of phosphorylated RSK1 Thr359.

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  • Ease-of-use Ease-of-use
  • Highly specific Highly specific
  • High sensitivity High sensitivity

This HTRF kit enables the cell-based quantitative detection of phosphorylated RSK1 Thr359.

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Overview

The Phospho-RSK1 (T359) Cellular kit is designed for the robust quantification of RSK1 phosphorylation at Threonine359 directly using a streamlined mix-and-read, no-wash protocol. RSK1 plays a pivotal role in cell proliferation and differentiation. This kit can be used from basic research to high throughput drug screening. Its versatility means it is suitable for many applications, ranging from basic research to the analysis of pharmacological questions in cellular models.

Benefits

  • COMPATIBLE WITH MANY CELL TYPES
  • SENSITIVE AND ROBUST
  • SPECIFICITY

Phospho-RSK1 (T359) Assay principle

The phospho-RSK1 assay measures RSK1 phosporylated at T59. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total RSK1 assay uses 2 labeled antibodies: one with a donor fluorophore, the other with an acceptor. The first antibody was 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.

Principle of the HTRF phospho-RSK1 (Thr359/Ser363) assay

Phospho-RSK1 (T359) 2-plate Assay Protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis. Then lysates are transferred into a 384-well low volume detection plate before the addition of HTRF Phospho-RSK1 (T359) detection reagents. This protocol enables the cells' viability and confluence to be monitored.

2-plate protocol of the HTRF phospho-RSK1 (Thr359/Ser363) assay

Phospho-RSK1 (T359) 1-plate assay protocol

Detection of Phospho-RSK1 (T359) 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.

1-plate protocol of the HTRF phospho-RSK1 (Thr359/Ser363) assay

Total and phospho RSK1 (T359) detection in EGF-stimulated A431 cells

A431 cells were plated under 100 µl in a 96-well plates (80,000 cells/well) in complete culture medium and incubated overnight at 37°C, 5% CO2. The day after, cells were treated with 50 µl of increasing concentrations of EGF for 5 min at 37°C, 5% CO2.

After medium removal, cells were then lysed with 100 µL of supplemented lysis buffer #2 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred into a low volume white microplate before the addition of 2 µL of the HTRF D2 detection reagent and 2 µL HTRF Eu-K detection reagent. Total RSK1 and phospho-RSK1 T359 were quantified in separate wells. The HTRF signal was recorded after ON incubation.

Validation of c-RAF kits on A431 cells

Function and regulation of RSK1

The Ribosomal protein S6 kinase alpha-1 (RSK1) is a downstream component of the MAP kinase and ERK signaling pathway and participates to the regulation of transcription factors, cell proliferation, survival, and differentiation with Serine/Threonine kinase.

It is phosphorylated at Thr359/Ser363 by MAPK1/ERK as an initial activation, and then autophosphorylates at Ser380 which allow PDK1 binding.

RSK1 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

Cisbio 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

HTRF assays for Oncology and Inflammation

Signaling in the immune system - Brochures

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

TCR signaling investigation with HTRF phospho assays

Study a pathway of interest in PBMC and T cells - Application Notes

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

An innate and adaptive immunity recap

Insight into the diversity of cells & signaling pathways - Guides

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

Plate Reader Requirement

Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.

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