Total cRaf Cellular Kit HTRF®
This HTRF kit enables the cell-based detection of total cRaf as a readout of the MAPK pathway.
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Low sample consumption
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High sensitivity
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All inclusive kit
Overview
The Total cRaf Cellular Assay kit is designed for the robust quantification of total cRaf 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
Total-cRaf assay principle
The Total cRaf assay quantifies the expression level of cRaf in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total cRaf assay uses two labeled antibodies, one coupled to a donor fluorophore and the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In the presence of eNOS in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein's expression under a no-wash assay format.
TotalcRaf two-plate assay protocol
The two-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 Total cRaf detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total cRaf one-plate assay protocol
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.
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.
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.
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 - Technical Notes
Optimize your HTRF cell signaling assays on tissues
HTRF and WB compatible guidelines - Technical Notes
Best practices for analyzing tumor xenografts with HTRF phospho assays
Protocol for tumor xenograft analysis with HTRF - Technical Notes
Key guidelines to successful cell signaling experiments
Mastering the art of cell signaling assays optimization - Guides
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
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
A single technology for 2D cells, 3D cells, and xenograft models - Posters
PI3K/AKT/mTor translational control pathway - 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
From 2D, 3D cell cultures to xenografts: A smart HTRF platform to maximize anticancer drug discovery
One technology across all samples - 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
HTRF Alpha-tubulin Housekeeping kit
Properly interpret your compound effect - Application Notes
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling
Analyse of PI3K/AKT/mTor translational control pathway - Application Notes
How to run a cell based phospho HTRF assay
What to expect at the bench - Videos
Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening
In collaboration with Bayer - Scientific Presentations
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
Product Insert cRaf Total Kit / 64CRAFTPEG-64CRAFTPEH
64CRAFTPEG-64CRAFTPEH - Product Insert
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
Safety Data Sheet (DEU) cRaf Total Kit / 64CRAFTPEG
64CRAFTPEG - Safety Data Sheet
Safety Data Sheet (ELL) cRaf Total Kit / 64CRAFTPEG
64CRAFTPEG - Safety Data Sheet
Safety Data Sheet (FRA-FR) cRaf Total Kit / 64CRAFTPEG
64CRAFTPEG - Safety Data Sheet
Safety Data Sheet (ITA) cRaf Total Kit / 64CRAFTPEG
64CRAFTPEG - Safety Data Sheet
Safety Data Sheet (SPA) cRaf Total Kit / 64CRAFTPEG
64CRAFTPEG - Safety Data Sheet
Safety Data Sheet (ENG-GB) cRaf Total Kit / 64CRAFTPEG
64CRAFTPEG - Safety Data Sheet
Safety Data Sheet (ENG-US) cRaf Total Kit / 64CRAFTPEG
64CRAFTPEG - Safety Data Sheet
Safety Data Sheet (DEU) cRaf Total Kit / 64CRAFTPEH
64CRAFTPEH - Safety Data Sheet
Safety Data Sheet (ELL) cRaf Total Kit / 64CRAFTPEH
64CRAFTPEH - Safety Data Sheet
Safety Data Sheet (FRA-FR) cRaf Total Kit / 64CRAFTPEH
64CRAFTPEH - Safety Data Sheet
Safety Data Sheet (ITA) cRaf Total Kit / 64CRAFTPEH
64CRAFTPEH - Safety Data Sheet
Safety Data Sheet (SPA) cRaf Total Kit / 64CRAFTPEH
64CRAFTPEH - Safety Data Sheet
Safety Data Sheet (ENG-GB) cRaf Total Kit / 64CRAFTPEH
64CRAFTPEH - Safety Data Sheet
Safety Data Sheet (ENG-US) cRaf Total Kit / 64CRAFTPEH
64CRAFTPEH - Safety Data Sheet
Plate Reader Requirement
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