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 enclosed in parentheses are predicted to react based on 100% sequence homology.
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.
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.
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.
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- Meloche, S. and Pouysségur, J. (2007) Oncogene 26, 3227-39.
- Roberts, P.J. and Der, C.J. (2007) Oncogene 26, 3291-310.
- Rubinfeld, H. and Seger, R. (2005) Mol Biotechnol 31, 151-74.
- Murphy, L.O. and Blenis, J. (2006) Trends Biochem Sci 31, 268-75.
- Dalby, K.N. et al. (1998) J Biol Chem 273, 1496-505.
- Marais, R. et al. (1993) Cell 73, 381-93.
- Kortenjann, M. et al. (1994) Mol Cell Biol 14, 4815-24.
- Owens, D.M. and Keyse, S.M. (2007) Oncogene 26, 3203-13.
- Emre, Y. et al. (2007) Biochem J 402, 271-8.
- Cuthbert, P.C. et al. (2007) J Neurosci 27, 2673-82.
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- 7177 PathScan® Phospho-p44/42 MAPK (Thr202/Tyr204) Sandwich ELISA Kit
- 7050 PathScan® Total p44/42 MAPK (Erk1/2) Sandwich ELISA Kit
- 4372 p44 MAP Kinase (Erk1) Antibody
- 4696 p44/42 MAPK (Erk1/2) (L34F12) Mouse mAb
- 4695 p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb
- 9102 p44/42 MAPK (Erk1/2) Antibody
- 4377 Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (197G2) Rabbit mAb
- 9106 Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (E10) Mouse mAb
- 9101 Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Antibody
- 7076 Anti-mouse IgG, HRP-linked Antibody
- 9803 Cell Lysis Buffer (10X)
- 7004 TMB Substrate
- 7002 STOP Solution
- 9808 Phosphate Buffered Saline (PBS-20X)
- 9809 Phosphate Buffered Saline with Tween 20 (PBST-20X)
For Research Use Only. Not For Use In Diagnostic Procedures.