Phospho-MEK1 (Ser218/222), Phospho-MEK1/2 (Ser218/222), Phospho-MEK1 (Ser298) & Total MEK1 Cellular Assay Kits

HTRF® cell-based assays for studying the RAS-RAF-MEK cascade in the MAPK/ERK pathway

Cisbio's cell-based homogeneous phospho-MEK1 & phosho-MEK1/2 assays enable a MAPK signaling pathway readout, by allowing the quantitative detection of the MAP2K1 kinase phosphorylated on serine 218/222 or on serine 298. 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.

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.

The buffers of both HTRF® phospho- and total MEK1 assays are compatible, enabling an analysis of the phosphorylated and the total protein populations from one lysate sample.

   Check our lysis buffer compatibility      Check our species compatibility

Assay principle

HTRF® - the homogeneous cell-based sandwich immunoassay

Cisbio Bioassay’s phospho- and total MEK1& MEK1/2 assays are based on a TR-FRET sandwich immunoassay format comprising two specific anti-MEK1 &1/2 antibodies, one labeled with Cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind with MEK1or MEK1/2, and the proximity of donor and acceptor then leads to a fluorescent TR-FRET signal. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.
The assays can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho- or total-protein can be quantitatively detected using the HTRF phospho- and total cellular assay kit reagents and most TR-FRET multimode plate readers.

A simpler, more flexible assay protocol – adapted to your applications

Two-plate assay protocol

For added flexibility, the assay can be run under a two-plate assay protocol, where cells are plated and treated in a 96-well culture plate. For detection, lysates are subsequently transferred to a 384-well small volume assay plate where the HTRF reagents are added. This also enables monitoring the cells' viability and confluence in an appropriate cell culture plate.

One-plate assay protocol

The protocol can be further streamlined to a one-plate assay in which plating, treating and detecting are all performed in a single plate. No wash steps are required. Ideal for HTS - this protocol means enhanced speed and simplicity, enabling all throughputs and fast results while maintaining high quality and sensitive output.

What to expect at the bench

This video covers the principles of phospho-HTRF assays and gives guidelines for performing them, using our phospho-ERK assay as an example.

Guidelines and tips to get the best out of your assays

Guidelines for cell culture and Lysis to prior detection, Download tehcnical note

Download our cell-based phospho-protein data normalization application note

Product performances

1. Western Blot versus HTRF assay

HeLa cells were grown in a T175 flask 37°C, 5% Co2, 2 days

Stimulation was done with EGF  2nM for 5 min. After removal of cell culture medium, 3ml of supplemented lysis buffer were added and incubated for 30 min. Soluble supernatants were collected after 10 min centrifuging. Equal amounts of lysates were used for a side by side comparison of WB and HTRF.

Both assays HTRF phospho-MEK1/2 and phospho-MEK1 shows the same level of sensitivity as Western Blot.

2. PMA dose-response on NIH 3T3 cells

Murine NIH 3T3 cells (25,000 cells/well) were stimulated for 15 minutes at 37°C with various concentrations of PMA. After a 30 minute lysis incubation time, phosphorylated MEK1 or MEK1/2 were measured using the two-plate assay protocol of the 2 assays. 

3. Inhibition effect of Erlotinib (an EGFR inhibitor) and of L779,450 (a Raf inhibitor) on HeLa cells

HeLa cells (50,000 cells/well) were incubated for 15 min at 37°C with various concentrations of the 2 inhibitors, Erlotinib (the EGFR inhibitor) and L777,450 (a Raf inhibitor). EGF 0.8nM (agonist) was then added and incubated for 10 minutes. After a 30 minute lysis incubation time, inhibition of the phosphorylated MEK was measured using the Phospho-MEK1/2 or the Phospho-MEK1 assays. Assays were done using the two-plate assay protocol.

5. Validation of the total-MEK1 assay on various species of cell lines: human, mouse and hamster

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, 3mL 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 16µL of cell lysate. Cell lines from other species have not been tested, hence they must be evaluated case by case.

6. 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.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
MEK1 phospho-S298 kit - 500 tests63ADK021PEG
MEK1 phospho-S298 kit - 10,000 tests63ADK021PEH
MEK1 phospho-S298 kit - 50,000 tests63ADK021PEY
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MEK1 phospho-S218/222 kit - 500 tests64ME1PEG
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MEK1 phospho-S218/222 kit - 10,000 tests64ME1PEH
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MEK1 phospho-S218/222 kit - 50,000 tests64ME1PEY
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MEK1/2 phospho-S218/222 kit - 500 tests64ME2PEG
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MEK1/2 phospho-S218/222 kit - 10,000 tests64ME2PEH
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MEK1/2 phospho-S218/222 kit - 50,000 tests64ME2PEY
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MEK1 total kit - 500 tests64NE1PEG
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MEK1 total kit - 10,000 tests64NE1PEH
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MEK1 total kit - 50,000 tests64NE1PEY
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Companion products

DescriptionCat. noProduct insertMSDS
MEK1 phospho-S298 kit - control lysate63ADK021TDA
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MEK1 phospho-S218/222 kit control lysate64ME1TDA
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MEK1/2 phospho-S218/222 kit control lysate64ME2TDA
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MEK1 total kit control lysate64NE1TDA
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