Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-p44 MAPK (Thr202/Tyr204) Sandwich ELISA Kit #7315

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Kit Includes Volume Solution Color
p44 MAPK Ab Coated Microwells
P-p44/42 MAPK (T202/ Y204) Detection Ab 11 ml Green
Anti-mouse IgG, HRP-linked Antibody 11 ml Red
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) #9803 15 ml Yellowish

Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H M

Reactivity Key:  H=Human  M=Mouse
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

CST’s PathScan® Phospho-p44 MAPK (Thr202/Tyr204) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of p44 MAP Kinase (Erk1) when phosphorylated at Thr202/Tyr204. A p44 MAP Kinase Rabbit Antibody has been coated onto the microwells. After incubation with cell lysates, p44 MAP Kinase (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a phospho-p44/42 MAPK (Thr202/Tyr204) Mouse Detection Antibody is added to detect phosphorylation of Thr202/Tyr204 on the captured p44 MAP Kinase (Erk1) protein. Anti-mouse IgG, HRP-linked antibody #7076 is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of absorbance for this developed color is proportional to the quantity of p44 MAP Kinase (Erk1) phosphorylated at Thr202/Tyr204.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

CST’s PathScan® Phospho-p44 MAPK (Thr202/Tyr204) Sandwich ELISA Kit #7315 detects endogenous levels of phospho-p44 MAP Kinase when phosphorylated at Thr202/Tyr204. As shown in Figure 1, a significant induction of p44 MAP Kinase (Erk1) phosphorylation at Thr202/Tyr204 can be detected in PDGF-treated NIH/3T3 cells using the Phospho-p44 MAPK (Thr202/Tyr204) Sandwich ELISA Kit #7315. The level of total p44 MAP Kinase (Erk1) detected by Western analysis remains unchanged. Note: This assay may have an increased absorbance reading if the diluted lysate that is applied to the microwells contains a final concentration of 0.5% sodium deoxycholate or 0.05% SDS. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

Western Blotting

Western Blotting

Figure 1. Treatment of NIH/3T3 cells with PDGF stimulates phosphorylation of p44 MAP Kinase (Erk1) at Thr202/Tyr204, detected by PathScan® Phospho-p44 MAPK (Thr202/Tyr204) Sandwich ELISA Kit #7315, but does not affect the level of total p44 MAP Kinase (Erk1) protein detected by Western analysis. NIH/3T3 cells (70-80% confluent) were starved overnight and treated with 100 ng/mL PDGF for 5 minutes at 37ºC. The absorbance readings at 450 nm are shown in the top figure, while the corresponding Western blots, using p44 MAP Kinase (Erk1) Antibody #4372 (left panel) and Phospho-p44/42 MAPK (Thr202/Tyr204) (E10) Mouse mAb #9106 (right panel), are shown in the bottom figure.

Sandwich ELISA

Sandwich ELISA

Figure 2. The relationship between the protein concentration of the lysate from untreated and PDGF-treated NIH/3T3 cells and the absorbance at 450 nm is shown.

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 research investigators consider it 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 or MAP3K), a MAP kinase kinase (MAPKK or MAP2K), and a MAP kinase (MAPK). Multiple p44/42 MAP3Ks have been identified, including members of the Raf family, as well as Mos and Tpl2/COT. MEK1 and MEK2 are the primary MAPKKs in this pathway (5,6). MEK1 and MEK2 activate p44 and p42 through phosphorylation of activation loop residues Thr202/Tyr204 and Thr185/Tyr187, respectively. Several downstream targets of p44/42 have been identified, including p90RSK (7) and the transcription factor Elk-1 (8,9). p44/42 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.

  1. Roux, P.P. and Blenis, J. (2004) Microbiol Mol Biol Rev 68, 320-44.
  2. Baccarini, M. (2005) FEBS Lett 579, 3271-7.
  3. Meloche, S. and Pouysségur, J. (2007) Oncogene 26, 3227-39.
  4. Roberts, P.J. and Der, C.J. (2007) Oncogene 26, 3291-310.
  5. Rubinfeld, H. and Seger, R. (2005) Mol Biotechnol 31, 151-74.
  6. Murphy, L.O. and Blenis, J. (2006) Trends Biochem Sci 31, 268-75.
  7. Dalby, K.N. et al. (1998) J Biol Chem 273, 1496-505.
  8. Marais, R. et al. (1993) Cell 73, 381-93.
  9. Kortenjann, M. et al. (1994) Mol Cell Biol 14, 4815-24.
  10. Owens, D.M. and Keyse, S.M. (2007) Oncogene 26, 3203-13.
  11. Emre, Y. et al. (2007) Biochem J 402, 271-8.
  12. Cuthbert, P.C. et al. (2007) J Neurosci 27, 2673-82.

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