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HTRF Human Total A-RAF Detection Kit, 10,000 assay points

Total A-RAF Product image
HTRF Human Total A-RAF Detection Kit, 10,000 assay points
Total A-RAF Product image
Total A-RAF Product image
Total A-RAF Product image
Total A-RAF Product image
Total A-RAF Product image

This HTRF kit allows for the cell-based quantitative detection of total A-RAF.

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Feature Specification
Application Cell Signaling
Sample Volume 16 µL
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This HTRF kit allows for the cell-based quantitative detection of total A-RAF.

View product information
Product Variants
Unit Size: 500 assay points
Part #:
64ARAFTPEG
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).
Total A-RAF Product image
Total A-RAF Product image
HTRF Human Total A-RAF Detection Kit, 10,000 assay points
Total A-RAF Product image
Total A-RAF Product image
Total A-RAF Product image
HTRF Human Total A-RAF Detection Kit, 10,000 assay points
Total A-RAF Product image
Total A-RAF Product image

Product information

Overview

A-RAF is a proto-oncogene protein kinase. The RAS-RAF-MEK signaling cascade plays a pivotal role in modulating cellular processes such as proliferation, differentiation, and survival. This pathway is often constitutively activated in human malignancies characterized by RAS or RAF oncogenic drivers. Developing chemotherapeutic cRaf inhibitors is an attractive area of research in oncology and infectious diseases.

Specifications

Application
Cell Signaling
Automation Compatible
Yes
Brand
HTRF
Detection Modality
HTRF
Lysis Buffer Compatibility
Lysis Buffer 1
Molecular Modification
Total
Product Group
Kit
Sample Volume
16 µL
Shipping Conditions
Shipped in Dry Ice
Target
A-RAF
Target Class
Phosphoproteins
Target Species
Human
Technology
TR-FRET
Therapeutic Area
Inflammation
Oncology
Unit Size
10,000 assay points

How it works

Total A-RAF assay principle

The Total A-RAF assay measures A-RAF levels in cells. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses 2 antibodies, one labeled with a donor fluorophore and the other with an acceptor. In presence of A-RAF this 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 total protein present in the sample and provides a means of assessing the protein's phosphorylation state under a no-wash assay format.

generic-R-D-attributes-assay-principle-total-without-phospho-1.png

 Total A-RAF two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of Total A-RAF HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

generic R D attributes assay protocol how it works 2 plates

 

Total A-RAF one-plate assay protocol

Detection of Total A-RAF 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 allows miniaturization while maintaining robust HTRF quality.

generic R D attributes assay protocol how it works 1 plate

Assay validation

Induction of Total A-RAF in cellular model and siRNA-treated cells

HeLa cells were plated at different cell densities at 100 µl in 96-well plates in complete culture medium. For siRNA Treated wells, siRNA in culture media was added (Final concentration: 60 nM Dharmacon ON-TARGETplus siRNA and 2% DharmaFECT transfection reagent). For No-siRNA-treated wells, 100 µl of media+transfection reagent were added. For Untreated wells, 100 µl of plain media were added. Cells were then incubated for 48hrs at 37°C, 5% CO2.

After incubation, the media were removed, and cells were lysed with 100 µL of lysis buffer #1 at 1X for 30 minutes at RT under gentle shaking. Next, 16 µL of lysate were transferred into a low volume white microplate before the addition of 2 µL of HTRF d2 detection reagent and 2 µL of HTRF Eu-K detection reagent. The HTRF signal was recorded after ON incubation.

assay validation siRNA a RAF Total 64ARAFTPEG 64ARAFTPEH 64ARAFTPEY

Simplified pathway

A-RAF signaling pathway

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

Simplified pathway a RAF 64ARAFTPEG 64ARAFTPEH 64ARAFTPEY

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