Total ERK cellular kit

This HTRF kit monitors the cellular ERK expression level and can be used as a normalization assay for the phospho-ERK kit.

Ease-of-use
High sensitivity
Highly specific

This HTRF kit monitors the cellular ERK expression level and can be used as a normalization assay for the phospho-ERK kit.

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Overview

The Total-ERK1/2 assay is ideal for ERK normalization with the phospho-ERK kits. It is compatible with the buffers from the phospho or Advanced phospho-ERK kits, so the same lysate can be used for fast and easy analysis of the total and the phosphorylated protein populations.

Benefits

COMPATIBLE WITH MANY CELL TYPES
SPECIFICITY
DATA NORMALIZATION

Total-ERK assay principle

The Total-ERK assay quantifies the expression level of ERK in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-ERK 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 ERK 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 proteins expression under a no-wash assay format.

Total-ERK 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 ERK HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total-ERK 1-plate assay protocol

Detection of total ERK 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.

Validation of total-ERK1/2 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. ERK1/2 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.

Validation of total-ERK assay on various cell lines

Total-ERK assay to control phosphorylation of ERK1/2

A431 cells (100,000 cells/well) were activated with EGF for 10 min, using the two-plate assay protocol of the Phospho-ERK1/2 and Total-ERK1/2 assays. As expected, results obtained show a dose-response increase of ERK1/2 phosphorylation upon EGF stimulation while ERK1/2 expression level remains constant.

dose-response increase of ERK1/2 phosphorylation upon EGF stimulation while ERK expression level remains constant.

MAPK/ERK cell signaling

The MAPK/ERK signaling cascade is activated by a wide variety of receptors involved in growth and differentiation. The core signal transduction cascade elicits regulation of numerous cellular processes including adhesion, migration, apoptosis, differentiation and metabolism. MEK1/2 catalyze the phosphorylation of ERK1/2 at Tyr204 and Thr202. In response, ERK phosphorylates hundreds of cytoplasmic and nuclear substrates. The wide complexity and diversity of MAPK signaling makes ERK a key regulator and major signaling node in biology.

ERK in GPCR signaling

ERK modulation plays a major role in GPCR signaling and is therefore an important measurable GPCR readout. GPCRs act via G proteins to regulate a wide range of cellular functions. Upon stimulation, these receptors activate effectors like adenylate cyclase and phospholipase C, which influence not only intracellular concentrations of second messengers like cAMP and Ca2+, but also mediate ERK1/2 phosphorylation. GPCRs have also been shown to mediate ERK1/2 activation in a G protein-independent but beta-arrestin dependent manner.

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Total ERK cellular kit

Overview

Benefits

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Product Insert

Product Insert ERK total Kit / 64NRKPEG

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Product Insert ERK total Kit / 64NRKPEG-64NRKPEH

Application Notes

Guides

Application Notes

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