采用Surefire AlphaScreen® 试剂盒((TGR Biosciences and PerkinElmer)进行pERK检测

Cassandra Koole; Denise Wootten; Patrick Sexton

doi:10.21769/BioProtoc.806

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Peer-reviewed

pERK Detection Assays Using the Surefire AlphaScreen^® Kit (TGR Biosciences and PerkinElmer)

采用Surefire AlphaScreen® 试剂盒((TGR Biosciences and PerkinElmer)进行pERK检测

CK Cassandra Koole email

DW Denise Wootten

PS Patrick Sexton

发布: 2013年07月05日第3卷第13期 DOI: 10.21769/BioProtoc.806 浏览次数: 10582

评审: Cheng Zhang

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参见作者原研究论文

The authors used this protocol in:

Cover of Proceedings of the National Academy of Sciences of the United States of America, featuring study using the protocol.

Nov 2012

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Abstract

Extracellular signal-regulated kinase 1 and 2 (ERK1/2) are serine/threonine protein kinases that are phosphorylated on Thr202/Tyr204 (ERK1) and Thr185/Tyr187 (ERK2). Phosphorylation of ERK1/2 (pERK1/2) arises from multiple stimuli, resulting in physiological responses that include cell growth, proliferation and differentiation. This protocol has been optimized for the detection of ligand-mediated pERK1/2 in adherent immortal cell lines expressing G protein-coupled receptors (GPCRs).

Materials and Reagents

Dulbecco’s modified eagle medium (DMEM) (Life Technologies, Gibco^®, catalog number: 11995-065 )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
SureFire^® Reagents (Includes Lysis, Activation and Reaction buffers) (TGR BioSciences, catalog number: TGRES500 )
AlphaScreen^® General IgG (Protein A) detection kit (PerkinElmer, catalog number: 6760617 )
White ProxiPlate 384-well microplate (PerkinElmer, catalog number: 6008280 )
TopSeal (PerkinElmer, catalog number: 6005250 )
NaCl
KCl
Na₂HPO₄
KH₂PO₄
Phosphate buffered saline (PBS) (see Recipes)
Detection buffer (see Recipes)

Equipment

Fusion-α plate reader or Envision plate reader with appropriate Alphascreen detection modules (PerkinElmer)
Sterile 96-well clear flat bottom plates (BD Biosciences, Falcon^®, catalog number: 353072 )
Humidified incubator
Multichannel pipettes
Micropipettes
Orbital shaker

Procedure

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Koole, C., Wootten, D. and Sexton, P. M. (2013). pERK Detection Assays Using the Surefire AlphaScreen^® Kit (TGR Biosciences and PerkinElmer). Bio-protocol 3(13): e806. DOI: 10.21769/BioProtoc.806.