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Cellul'erk for direct detection of Phosphorylated ERK1/2 in cell-based format

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 that influence intracellular concentrations of second messengers (cyclic AMP, diacylglycerol, inositol 1,4,5 trisphosphate and Ca2+) but also mediate Extracellular Signal Regulated kinase (ERK1/2) phosphorylation. Activation of Gαi/o, Gαs, Gαq/11 or Gα12/13 modulates ERK1/2 activation via numerous mechanisms. In addition, both α and βγsubunits of G proteins can stimulate ERK1/2 phosphorylation through transactivation of receptor tyrosine kinases (RTKs). GPCRs have also been shown to mediate ERK1/2 activation in a G protein-independent but β-arrestin dependent manner.

Cisbio Bioassays has developed Cellul'erk, a highly accurate HTRF assay for measuring phosphorylated ERK1/2 in 96-, 384- and 1536-well formats providing for an HTS compatible assay.

Features:
  • Simple assay protocols designed
    • for research studies (96-well plates)
    • for HTS (384-small vol. plates)
  • Cell-based functional assay
  • Antibody-based detection
  • Direct detection
Applications:
  • Gi, Gs, or Gq coupled receptor activation
  • Mitogen-activated protein kinase (MAPK) pathway (RTKs activity)
  • β -Arrestin signaling

Assay Principle:

 

Assay can be run with cell lysates or using whole cells upon GPCR or RTK activation, ERK1/2 is phosphorylated, after lysis of the cell membrane, phosphorylated ERK1/2 can be detect using the kit reagents.
Cellul'erk is based on a sandwich immunoassay using an anti-phospho-ERK1/2 antibody labeled with d2 and an anti-ERK1/2 antibody labeled with Eu3+-cryptate. These antibodies may be pre-mixed and added in a single dispensing.

The assay can be run under a two-plate protocol. It 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 by HTRF® reagents.
This protocol allows the cells' viability and confluence to be monitored.

One-plate assay protocol:

Detection of phosphorylated ERK 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. 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




2. Screening robustness:

CHO-M1 (50,000 cells/well - 384-well small volume plate) are stimulated with Carbachol for 10 minutes at RT.
Z' values in fifty replicates are calculated between Unstimulated cells (basal level) and 2 selected Carbachol concentrations:1.3 μM and 4 μM corresponding to the EC80and EC100 respectively. Assay is performed using the One-plate assay protocol on a robotic system from CyBio™ (CyBi®vario equipped with 384/25μL pipeting head). Results are read on PheraStarplus (BMGLabtech).




3. Endogeneous receptor activation

Results obtained on HEK (50,000 cells) parental cell line activated with EGF for 10' after a 2 day serum starvation, using the two- plate assay protocol




4. Mitogen-activated protein kinase (MAPK) pathway

Results obtained on CHO-M1 (50,000 cells) activated with a direct protein kinase C (PKC) activator: phorbol 12-myristate 13-acetate (PMA) for 10'




Ordering Info:

 

Cellul'erk Reagents:

Size* Cat no.** Product Insert
200 tests 64ERKPEF upcoming
500 tests 64ERKPEG upcoming
10,000 tests 64ERKPEH upcoming
50,000 tests 64ERKPEI upcoming

* based on 20μL assay volume
** upon request

Companion Products:

Description Size* Cat no.
Erk_Blocking reagent 0.82mL 64EBRAAC
Erk_Lysis buffer 50mL 64CL7FDF


For more information or To Place an Order:
 

Europe and other countries
+33(0)466 796 705

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