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HTRF Human & Mouse Phospho-RSK1 (Ser380) Detection Kit, 10,000 Assay Points

This HTRF kit enables the cell-based quantitative detection of phosphorylated RSK1 Serine 380.

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption and disposal requirements under European REACH regulations (EC 1907/2006).

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Product Variants
Part number: 64RSK1S0PEG
Unit Size: 500 Assay Points
List price: USD 2,147.00
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USD 0.00
USD 2,147.00 /each
Part number: 64RSK1S0PEH
Unit Size: 10,000 Assay Points
List price: USD 12,490.00
Your price: Login to view
USD 12,490.00
USD 12,490.00 /each

Overview

The Phospho-RSK1 (S380) Cellular Kit is designed for the robust quantification of phosphorylated RSK1 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.

Specifications

Application
Cell Signaling
Assay Target Class
Phosphoproteins
Assay Technology
TR-FRET
Automation Compatible
Yes
Brand
HTRF
Detection Method
HTRF
Molecular Modification
Phosphorylation
Product Group
Kit
Sample Volume
16 µL
Shipping Conditions
Shipped in Dry Ice
Therapeutic Area
Infectious Diseases
Metabolism/Diabetes
Oncology & Inflammation
Rare Diseases
Unit Size
10,000 Assay Points

Video gallery

How it works

Phospho-RSK1 (S380) assay principle

The Phospho-RSK1 (S380) Assay measures RSK1 phosphorylation at Serine 380. 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, and 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.

rsk1-phospho-s380-assay-principle

 

Phospho-RSK1 (S380) 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 (S380) detection reagents. This protocol enables the cells' viability and confluence to be monitored.

rsk1-phospho-s380-assay-protocol-01

 

Phospho-RSK1 (S380) 1-plate assay protocol

Detection of Phospho-RSK1 (S380) 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.

rsk1-phospho-s380-assay-protocol-02

 

Assay validation

Total and phospho RSK1 (S380) 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 S380 were quantified in separate wells. The HTRF signal was recorded after ON incubation.

assay-validation-rsk1-phospho-s380

 

Simplified pathway

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.

pathway-rsk1-phospho-s380-64rsk1s0peg

 

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