HTRF® Cellular Kinase Assays
for studying phospho-AKT and phospho-ERK
Based on our homogeneous and robust HTRF assays, Cellul'erk, HTRF phospho-Akt (Ser473) and HTRF phospho-Akt (Thr308) assays are designed for detecting and studying activated Erk1/2 and Akt directly in whole cells. Using streamlined protocols, amenable to low-volume format, the kits are precisely designed for high-throughput screening.
Features:- Simple assay protocols: an alternative to Western Blot or ELISA
- Cell-based functional assays
- Antibody-based detection
- Homogeneous and direct detection
- HTRF robustness, sensitivity and reliability
- Suitable for exploring endogeneous and over-expressed receptors
- Use in many types of cells including primary
- All inclusive: all reagents in one kit
- AKT & MAPK Signaling pathways
- Cell surface receptors investigation
- AKT & ERK targeting
Cellular kinases assays available:
- Phospho Akt (Thr308)
- Phospho Akt (Ser473)
- Cellul'erk (Phospho erk1/2)
More assays are under development or can be optimized through our custom development service offer.
Assay Principle:
Assays can be run with cell lysates or using whole cells. Upon receptor activation, the kinases are activated and after the lysis of the cell membrane, phosphorylated kinases can be detect using the kit reagents. The assays are based on a sandwich immunoassay involving two monoclonal antibodies: anti-kinase antibody labeled with d2 and an anti-phospho-kinase antibody labeled with Eu 3+-cryptate. The antibodies may be pre-mixed and added in a single dispensing step.
All assays can be run under a two-plate protocol. They can also be further streamlined to a one-step assay.
Two-plate assay protocol:
Cells are plated, stimulated and next lysed in the same 96-well culture plate. Lysates are then transferred to the assay plate for the detection of phosphorylated ERK or Akt by HTRF reagents. This protocol allows the cells' viability and confluence to be monitored.
One-plate assay protocol:
Detection of phosphorylated ERK or AKT with HTRF reagents is performed in a single plate used for plating, stimulation and lysis. No washing steps are required. This protocol, HTS designed, allows miniaturization while maintaining HTRF quality.
Assay Performance:
1. Phospho-Akt (Thr308) assay IGF dose-response on CHO-M1 cells
Results obtained on CHO cells plated using 2 cell densities (25,000 - 50,000 cells per well), after been starved for 24 hours cells were stimulated for 10 minutes with various concentration of IGF. Phosphorylated Akt on Thr308 was measured after 3 hours 1/2 using the two-plate assay protocol
2. Phospho-Akt (Ser473) assay IGF dose-response on HEK 293 cells
Results obtained on HEK cells plated using 3 cell densities (12,500 - 25,000 - 50,000 cells per well), after been starved for 24 hours cells were stimulated for 10 minutes with various concentration of IGF. Phosphorylated Akt was measured after 3 hours 1/2 using the two-plate assay protocol.
3. Phospho-Akt (Ser473) assay and Cellul'erk growth factor stimulation time course on mouse cardiomyocyte cell line HL-1
Cells plated at 50,000 cells per well were starved for 16 hours and stimulated at room temperature for 3 different incubation times: 3, 7 and 15 min with various concentrations of growth factor based ligand stimulator.
Stimulation curves shown here were obtained using the two-plate assay protocol of the Cellul'erk assay for the assessment of phosphorylated Erk (fig. A) and the two-plate assay protocol of the Phospho-Akt assay for the study of phosphorylated Akt (fig.B)
Results used by permission of Acorda Therapeutics, 15 Skyline Drive, Hawthorne NY 10532 United States.
4. Cellul'erk assay Gαq/i coupled receptor activation:
Results obtained on CHO-CCR5 (25,000 cells) activated with Rantes & MIP-Iβ for 10', using the two-plate assay protocol.
Phosphorylation of Erk1/2 was measured using Cellul'erk assay.
Ordering Info:
Cellular kinases reagents:
| Product name | Cat no. | Size* | Product Insert | MSDS |
| Cellul'erk (phospho-Erk1/2) | 64ERKPEG | 500 tests | ||
| Cellul'erk (phospho-Erk1/2) | 64ERKPEH | 10,000 tests | ||
| Cellul'erk (phospho-Erk1/2) | 64ERKPEI | 50,000 tests | - | |
| Phospho-Akt (Ser473) | 64AKSPEG | 500 tests | ||
| Phospho-Akt (Ser473) | 64AKSPEH | 10,000 tests | ||
| Phospho-Akt (Ser473) | 64AKSPEI | 50,000 tests | ||
| Phospho-Akt (Thr308) | 64AKTPEG | 500 tests | ||
| Phospho-Akt (Thr308) | 64AKTPEH | 10,000 tests | ||
| Phospho-Akt (Thr308) | 64AKTPEI | 50,000 tests | - |
Companion Products:
| Product name | Cat no. | Size |
| Cellular kinase blocking reagent | 64KB1AAC | 6 mL. |
| Cellular kinase lysis buffer | 64KL1FDF | 130 mL |
* based on 20μL assay volume
For more information or To Place an Order:
Europe and other countries
+33(0)466 796 705
