Product Pathways - MAPK Signaling

p44/42 MAP Kinase Assay Kit (Nonradioactive) #9800

Kit Includes	Quantity	Source
Immobilized Phospho-p44/42 MAPK (Thr202/Tyr204) Mouse mAb # 9109	600 microliters	Mouse
Phospho-Elk-1 (Ser383) Antibody # 9181	100 microliters	Rabbit
Elk-1 Fusion Protein # 9184	80 micrograms
Kinase Buffer (10X) # 9802	15 milliliters
Cell Lysis Buffer (10X) # 9803	15 milliliters
ATP (10 mM) # 9804	50 microliters
Anti-rabbit IgG, HRP-linked Antibody # 7074	50 microliters	Goat
Anti-biotin, HRP-linked Antibody # 7075	100 microliters	Goat
Biotinylated Protein Ladder Detection Pack # 7727	100 microliters
20X LumiGLO^® Reagent and 20X Peroxide # 7003	5 milliliters each
Active p42 MAP Kinase (10 ng/?l)	10 microliters

Molecular Weight

Reactivity

H M R

Reactivity Key: H=Human M=Mouse R=Rat

Description

Nonradioactive p44/42 MAP Kinase Assay Kit provides all the reagents necessary for measuring p44/42 MAP kinase activity in the cell. Immobilized Phospho-p44/42 MAPK (Thr202/Tyr204) mAb is used to immunoprecipitate active p44/42 MAP kinase from cell extracts, then an in vitro kinase assay is performed using Elk-1 protein as a substrate. Elk-1 phosphorylation is then detected by Western blotting using Phospho-Elk-1 (Ser383) Antibody.Improvements Over Conventional Assays:Improved sensitivity without radioactivityPhospho-specific antibodies allow site-specific analysisDramatically increased signal to noise ratio over conventional IP/kinase assaysLow background activityIncludes complete system to assay kinase activity

Western Blotting

p44/42 MAP Kinase-induced phosphorylation of Elk-1 was measured by quantitative immunoblotting with Phospho-Elk-1 Antibody (A) and compared to direct measurement of phosphate incorporation using [γ-³²P]-ATP (B). (Note exposure time difference).

Improvements Over Conventional Assays

No Radioactivity: Improved sensitivity without radioactivity.
Specificity: Phospho-specific antibodies allow site-specific analysis.
Signal to Noise: Dramatically increased signal to noise ratio over conventional IP/kinase assays.
Low Background Activity
Complete System: Includes everything needed to assay kinase activity.

Background

Mitogen-activated protein kinases (MAPKs) are a widely conserved family of serine/threonine protein kinases involved in many cellular programs such as cell proliferation, differentiation, motility, and death. The p44/42 MAPK (ERK1/2) signaling pathway can be activated in response to a diverse range of extracellular stimuli including mitogens, growth factors, and cytokines (1-3) and is an important target in the diagnosis and treatment of cancer (4). Upon stimulation, a sequential three-part protein kinase cascade is initiated, consisting of a MAP kinase kinase kinase (MAPKKK), a MAP kinase kinase (MAPKK), and a MAP kinase. While multiple ERK1/2 MAP3Ks have been identified, including the Raf family, Mos, and Tpl2/Cot, MEK1 and MEK2 are the primary MAPKKs in this pathway (5,6). MEK1 and MEK2 activate ERK1/p44 and ERK2/p42 through phosphorylation of activation loop residues Thr202/Tyr204 and Thr185/Tyr187, respectively. Several downstream targets of ERK1/2 have been identified, including p90RSK (7) and the transcription factor Elk-1 (8,9). ERK1/2 are negatively regulated by a family of dual-specificity (Thr/Tyr) MAPK phosphatases, known as DUSPs or MKPs (10), along with MEK inhibitors such as U0126 and PD98059.

Application References

Russell, K.S. et al. (2000) Proc Natl Acad Sci U S A 97, 5930-5. This article references the use of p44/42 MAP Kinase Assay Kit (Nonradioactive) in the following applications: In vitro kinase assay
Tan, J. et al. (2000) J Neurosci 20, 7587-94. This article references the use of p44/42 MAP Kinase Assay Kit (Nonradioactive) in the following applications: In vitro kinase assay
Jost, M. et al. (2001) J Biol Chem 276, 6320-6. This article references the use of p44/42 MAP Kinase Assay Kit (Nonradioactive) in the following applications: In vitro kinase assay
Skapenko, A. et al. (2001) J Immunol 166, 4283-92. This article references the use of p44/42 MAP Kinase Assay Kit (Nonradioactive) in the following applications: In vitro kinase assay
Gui, Y. and Zheng, X.L. (2003) J Biol Chem 278, 53017-25. This article references the use of p44/42 MAP Kinase Assay Kit (Nonradioactive) in the following applications: In vitro kinase assay
Hsu, H.Y. et al. (2004) J Immunol 173, 5989-99. This article references the use of p44/42 MAP Kinase Assay Kit (Nonradioactive) in the following applications: In vitro kinase assay
Wong, C.H. et al. (2005) Endocrinology 146, 1192-204. This article references the use of p44/42 MAP Kinase Assay Kit (Nonradioactive) in the following applications: In vitro kinase assay
He, Z. et al. (2005) J Biol Chem 280, 2446-54. This article references the use of p44/42 MAP Kinase Assay Kit (Nonradioactive) in the following applications: In vitro kinase assay
Schlezinger, J.J. et al. (2006) Toxicol Sci 92, 433-44. This article references the use of p44/42 MAP Kinase Assay Kit (Nonradioactive) in the following applications: In vitro kinase assay
Jia, W. et al. (2006) Mol Cancer Res 4, 549-62. This article references the use of p44/42 MAP Kinase Assay Kit (Nonradioactive) in the following applications: In vitro kinase assay
Zampieri, C.A. et al. (2007) J Virol 81, 1230-40. This article references the use of p44/42 MAP Kinase Assay Kit (Nonradioactive) in the following applications: In vitro kinase assay
Ha, U. et al. (2007) J Immunol 178, 1736-47. This article references the use of p44/42 MAP Kinase Assay Kit (Nonradioactive) in the following applications: In vitro kinase assay

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