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HTRF Human Total cRaf Detection Kit, 500 Assay Points

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

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 & disposal requirements under European REACH regulations (EC 1907/2006).
Product Variants
Part number: 64CRAFTPEG
Unit Size: 500 Assay Points
List price: USD 2,147.00
Your price:
USD 2,147.00
USD 2,147.00 /each
Part number: 64CRAFTPEH
Unit Size: 10,000 Assay Points
List price: USD 12,490.00
Your price:
USD 0.00
USD 12,490.00 /each

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.

Specifications

Application
Protein Detection
Assay Points
500
Assay Target Type
Kit
Assay Technology
HTRF
Automation Compatible
Yes
Brand
HTRF
Detection Method
HTRF
Experimental Type
In vitro
Quantity
1
Shipping Conditions
Shipped in Dry Ice
Therapeutic Area
Cardiovascular
Metabolism/Diabetes
Oncology & Inflammation
Unit Size
500 Assay Points

Video gallery

How it works

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.

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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.

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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.

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Assay validation

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-cRaf-cellular-kit

 

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.

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Simplified pathway

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.

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Resources

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Guide
HTRF solutions, guide to major applications

This guide provides you an overview of HTRF applications in several therapeutic areas.

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SDS, COAs, Manuals and more

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