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HTRF Human Phospho-MEK1 (Ser298) Detection Kit, 500 Assay points
This HTRF kit enables the cell-based quantitative detection of MEK1 phosphorylation at Ser298.
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
Product information
Overview
The Phospho-MEK1 (Ser298) kit measures endogenous MEK1 protein phophorylated at Ser298, to help studies of the RAS-RAF-MEK cascade in the MAPK/ERK pathway. Phosphorylated by RAF, this dual specificity mitogen-activated protein kinase is responsible for the phosphorylation of ERK, one of the most important signaling nodes, and protein kinase in cell proliferation, survival, and differentiation pathways.
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
|
| Therapeutic Area |
Cardiovascular
Neuroscience
Oncology & Inflammation
|
| Unit Size |
500 Assay Points
|
Video gallery
How it works
Phospho-MEK1 (Ser298) assay principle
The Phospho-MEK1 (Ser298) assay measures MEK1 when phosphorylated at Ser298. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Phospho-MEK1 (Ser298) assay uses two antibodies: one labeled with a donor fluorophore, the other one with an acceptor. The first antibody was selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent 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.
Phospho-MEK1 (Ser298) 2-plate assay protocol
The 2 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-MEK1 (Ser298) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-MEK1 (Ser298) 1-plate assay protocol
Detection of Phosphorylated MEK1 (Ser298) 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.
Assay validation
Detection of phospho MEK1 (Ser298) on Hela cells
Hela cells were cultured in a T175 flask. At 80% of confluency, the cell culture medium was removed and 3mL of complete lysis buffer (1X) were added. The lysis step was done under gentle shaking for 30 minutes. Lysate was then collected and diluted. 16µL of each dilution were transferred into a 384-well plate for the detection of phospho MEK1 (Ser298).
Resources
Guide
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Brochure
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SDS, COAs, Manuals and more
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