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Phospho-cRaf (Ser43) Cellular Kit HTRF®

This HTRF kit enables the cell-based quantitative detection of phosphorylated c-RAF Ser43.
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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 c-RAF Ser43.
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Overview

The Phospho-c-RAF (Ser43) Cellular Assay kit is designed for the robust quantification of phoshorylated cRaf at Ser-43 directly using a streamlined mix-and-read, no-wash protocol. cRaf 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, and has applications in oncology and infectious disease research.

Benefits

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

Phospho-c-RAF (Ser43) Assay principle

The Phospho-c-RAF (Ser43) assay measures c-RAF when phosphorylated at Ser43. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Phospho-c-RAF (Ser43) 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.

Principle of the HTRF phospho-c-RAF (Ser43) assay

Phospho-c-RAF (Ser43) 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 Phospho-c-RAF (Ser43) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

2-plate protocol of the HTRF phospho-c-RAF (Ser43) assay

Phospho-c-RAF (Ser43) 1-plate assay protocol

Detection of Phosphorylated c-RAF (Ser43) 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-c-RAF (Ser43) assay

Total and phospho c-RAF (S43) detection in TPA-stimulated HeLa cells

HeLa cells were plated under 100 µl in a 96-well plates (100,000 cells/well) in complete culture medium and incubated overnight at 37°C, 5% CO2. The day after, medium was removed and 50 µl of serum-free culture medium was added to starve cells for 5h at 37°C, 5% CO2. Then cells were treated with 50 µl of increasing concentrations of TPA for 15 min at 37°C, 5% CO2.

After medium removal, cells were then lysed with 50 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 4 µL of the premixed HTRF phospho-cRaf (Ser43) or Total-cRaf detection reagents. The HTRF signal was recorded after ON incubation.

Validation of c-RAF kits on HeLa cells

Total and phospho c-RAF (S43) detection in TPA-stimulated HEK293T cells

HEK293T cells were plated under 100 µl in a 96-well plates (100,000 cells/well) in complete culture medium and incubated overnight at 37°C, 5% CO2. The day after, medium was removed and 50 µl of serum-free culture medium was added to starve cells for 5h at 37°C, 5% CO2. Then cells were treated with 50 µl of increasing concentrations of TPA for 15 min at 37°C, 5% CO2.

After medium removal, cells were then lysed with 50 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 4 µL of the premixed HTRF phospho-cRaf (Ser43) or Total-cRaf detection reagents. The HTRF signal was recorded after ON incubation.

Validation of c-RAF kits on HEK293T cells

Function and regulation of RSK1

RAF proto-oncogene serine/threonine-protein kinase (c-RAF) is par of the MAP kinases pathway where it links the upstream effector Ras to the downstream MAPK/ERK cascade. It therefore pays roles in cell fate decisions including proliferation, differentiation, apoptosis, survival and oncogenic transformation.

c-RAF is activated by Ser338 phosphorylation as a direct or indirect effect of PKC, or by GTP-bound Ras directly. Inactivation is ensured by the phosphorylation at Ser43 via MAPK/ERK-dependent feedback.

c-RAF signaling pathway

HTRF cellular phospho-protein assays

physiologically relevant results fo fast flowing research - Flyers

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

Insider Tips for successful sample treatment - Notes techniques

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Notes techniques

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Notes techniques

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF® cell signaling platform combined with iCell® Hepatocytes

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

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - 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 - Notes d'application

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Notes d'application

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

Increased flexibility of phospho-assays - Notes d'application

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Notes d'application

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

Analyse of PI3K/AKT/mTor translational control pathway - Notes d'application

How to run a cell based phospho HTRF assay

What to expect at the bench - Vidéos

Unleash the potential of your phosphorylation research with HTRF

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

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Vidéos

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

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

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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