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HTRF Phospho-c-RAF (Ser43) Detection Kit, 500 Assay Points

This HTRF kit allows for the cell-based quantitative detection of c-RAF when phosphorylated at Ser43.

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).

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Feature Specification
Application Cell Signaling
Sample Volume 16 µL

This HTRF kit allows for the cell-based quantitative detection of c-RAF when phosphorylated at Ser43.

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).

Click to copy promo code to clipboard.
SAVE 15% NOW on online orders. Use promo code below.
YEAREND15
Offer valid until 12/15. Terms and conditions apply.
Product Variants
Unit Size: 500 Assay Points
Part #:
64CRAFS43V2PEG
List Price
USD 2,147.00
Unit Size: 10,000 Assay Points
Part #:
64CRAFS43V2PEH
List Price
USD 12,490.00

Overview

cRaf is a proto-oncogene serine/threonine-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 disease.

Specifications

Application
Cell Signaling
Brand
HTRF
Buffer/Solvent
Lysis Buffer 2
Detection Modality
HTRF
Host Species
Human
Lysis Buffer Compatibility
Lysis Buffer 1
Lysis Buffer 2
Molecular Modification
Phosphorylation
Product Group
Kit
Sample Volume
16 µL
Shipping Conditions
Shipped in Dry Ice
Target
cRAF
Target Class
Phosphoproteins
Technology
TR-FRET
Therapeutic Area
Inflammation
Oncology
Unit Size
500 Assay Points

How it works

Phospho-cRAF (Ser43) assay principle

The Phospho-cRAF (Ser43) assay measures cRAF when phosphorylated at Ser43. 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. The first antibody was selected for its specific binding to the phosphorylated motif on the protein, and the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation 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 phosphorylated protein present in the sample and provides a means of assessing the protein's phosphorylation state under a no-wash assay format.

Principle of the HTRF Phospho Y1604 ALK assay
Phospho-cRAF (Ser43) 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 Phospho-cRAF (Ser43) HTRF detection reagents. This protocol allows for the cells' viability and confluence to be monitored.

Two-plate protocol of the HTRF Phospho Y1604 ALK assay
Phospho-cRAF (Ser43) one-plate assay protocol

Detection of Phosphorylated cRAF (Ser43) 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 facilitates miniaturization while maintaining HTRF quality.

One-plate protocol of the HTRF Phospho Y1604 ALK assay

Assay validation

Induction of phospho-cRAF (Ser43) in Hela cells

Hela cells were seeded in a 96-well culture-treated plate (100,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. The cells were treated for 15 minutes with increasing concentrations of phorbol 12-myristate 13-acetate (PMA).

After treatment, the cells were lysed with 50 µL of supplemented lysis buffer #2 for 30 minutes at RT under gentle shaking. For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Phospho-cRAF (Ser43) or Total cRAF ( # 64CRAFTPEG/H/Y) detection reagents were added. The HTRF signal was recorded after an overnight incubation. In parallel, the cell viability was assessed. To this end, 5 µL of the same lysate were transferred in an HTRF 96-well low volume white plate (# 66PL96005/025/100) and 25 µL of ATPlite 1step detection reagent were added (# 6016736/1/9). The luminescence signal was measured after a 10-min incubation in the dark at RT.

As expected, PMA triggered a dose-dependent increase of Phospho-cRAF (Ser43). In addition, the compound does not affect the cell viability as measured by cell viability indicator ATPLite.

assay validation
assay validation
Specificity of phospho-cRAF (Ser43) assay using HAP-1 KO cell lines

The Phospho-cRAF (Ser43) protein levels were assessed with the HTRF Phospho-cRAF Ser43 kit in HAP1 cells (WT) and different HAP1 cell lines Knocked-Out for cRAF, ARAF and BRAF.
The different cell lines were plated in a 96-well plate (100,000 cells/well) and cultured for 24h. The cells were then stimulated with PMA (20µM, 15 min). After treatment, the cells were lysed with 50 µL of supplemented lysis buffer #2 for 30 minutes at RT under gentle shaking.

assay validation

For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF phospho-cRAF (Ser43) detection reagents were added. An additional 4 µL of lysates (supplemented with 12 µL diluent #8) was also transferred into the microplate to monitor the alpha-tubulin level using the HTRF Alpha-tubulin Housekeeping Cellular Kit (64ATUBPEG/H). HTRF signal was recorded after an overnight incubation.

In HAP1-cRAF KO cells, the HTRF signal was equivalent to the non-specific signal (dotted line), indicating a complete cRAF gene silencing, whereas the phospho-cRAF (Ser43) level was well detected in the other cell line, as expected.

Revvity's catalog reference: HAP1-cRAF KO #HZGHC002807c010, HAP1-ARAF KO #HZGHC002806c004 and HAP1-BRAF KO #HZGHC000002c001

HTRF phospho-cRAF (Ser43) assay compared to Western Blot

HEK293 cells were grown in a T175 flask in complete culture medium at 37°C - 5% CO2 until 80% confluence. Cells were PMA stimulated (20µM, 15min) and lysed with 3 mL of supplemented lysis buffer#2 (1X) for 30 minutes at RT under gentle shaking.

assay validation

Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF Phospho-cRAF (Ser43) detection reagents. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

Using the HTRF phospho-cRAF (Ser43) assay, 500 cells/well were enough to detect a significant signal, while 2000 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore, in these conditions, the HTRF Phospho-cRAF (Ser43) assay was 4 times more sensitive than the Western Blot technique.

Simplified pathway

cRAF signaling pathway

RAF proto-oncogene serine/threonine-protein kinase (c-RAF) is part of the MAP kinases 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.

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.

Simplified pathway

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