Total cRaf Cellular Kit HTRF®

Associated products

This HTRF kit enables the cell-based detection of total cRaf as a readout of the MAPK pathway.

Low sample consumption
High sensitivity
All inclusive kit

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This HTRF kit enables the cell-based detection of total cRaf as a readout of the MAPK pathway.

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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.

2-plate protocol of the HTRF total-c-RAF assay

Total cRaf one-plate assay protocol

Detection of Total eNOS 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 total-c-RAF 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.

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.

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.

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

HTRF® phospho-protein platform facilitates the dissection of signaling pathways involved in insulin resistance and metabolic disorders

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

Implement HTRF phospho-protein assays at every step of the drug discovery process, from in vitro to in vivo models

A single technology for 2D cells, 3D cells, and xenograft models - Posters

Simplify complex pathway dissection by combining the power of HTRF® cellular phospho-assays and the flexibility of the CyBi®-FeliX liquid handling system

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