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HTRF Human Total MEK1 Detection Kit, 500 Assay Points

The total MEK1 kit quantifies cellular endogenous MEK1 when studying the MAPK/ERK pathway, and can be used as a normalization assay for the phospho-MEK1 kit.

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

The total MEK1 kit quantifies cellular endogenous MEK1 when studying the MAPK/ERK pathway, and can be used as a normalization assay for the phospho-MEK1 kit.

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 #:
64NE1PEG
List Price
USD 2,147.00
Unit Size: 10,000 Assay Points
Part #:
64NE1PEH
List Price
USD 12,490.00

Overview

The HTRF total MEK1 assay kit is designed for the quantitative detection of total MEK1, phosphorylated and unphosphorylated, to normalize the phosphorylation status of MEK1. It is the ideal assay for studying 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
Cell Signaling
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 Class
Phosphoproteins
Target Species
Human
Technology
TR-FRET
Therapeutic Area
Cardiovascular
Neuroscience
Oncology & Inflammation
Unit Size
500 Assay Points

Video gallery

How it works

Total-MEK1 assay principle

The Total-MEK1 assay quantifies the expression level of MEK1 in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-MEK1 assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of MEK1 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.

phospho-how-it-works-total-mek1-assay-principle

 

Total-MEK1 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding Total MEK1 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

phospho-how-it-works-total-mek1-2-plate-assay-protocol
 
 Total-MEK-1-plate assay protocol

Detection of total MEK 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.

phospho-how-it-works-total-mek-1-plate-assay-protocol

 

Assay validation

Validation of the total-MEK1 assay on various cell lines

Human (Hela, A431, HEK293, Jurkat), murine (NIH 3T3) and Hamster (CHO-K1) cells were grown in a T175 flask at 37°C, 5% CO2 for 2 days. After removal of cell culture medium, 3 mL of supplemented lysis buffer 3 were added and incubated for 45 min. Soluble supernatants were collected after 10 min centrifuging, total MEK1 was detected using 16 µL of cell lysate. Cell lines from other species have not been tested, hence they must be evaluated case by case.

assay-validation-mek1-total-1

 

Total-MEK1 assay used to control the phosphorylation status of MEK1

Hela cells (100,000 cells/well) were activated with EGF for 5 min, using the two-plate assay protocol of the Phospho-MEK1 (Ser218/222) and Total-MEK1 assays. Results obtained show a dose-response increase of MEK1 phosphorylation upon EGF stimulation while MEK1 expression level remains constant.

assay-validation-mek1-total-2

 

Resources

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