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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 Low sample consumption
  • High sensitivity High sensitivity
  • All inclusive kit All inclusive kit

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.

Principle of the HTRF Total-c-RAF assay

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.

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

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Product Insert cRaf Total Kit / 64CRAFTPEG-64CRAFTPEH

64CRAFTPEG-64CRAFTPEH - Product Insert

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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