Figure 1. Treatment of NIH/3T3 cells with PDGF stimulates phosphorylation of p44/42 MAPK at Thr202/Tyr204, detected by the PathScan® Phospho-p44/42 MAPK (Thr202/Tyr204) Sandwich ELISA Kit #7177, but does not affect the level of p44/42 MAPK detected by western analysis. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots, using p44/42 MAPK (Erk1/2) Antibody #9102 (left) and Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370 (right), are shown in the bottom figure.
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.
|REACTIVITY||H M R|
|Product Includes||Volume||Solution Color|
|Phospho-p44/42 MAPK (T202/Y204) Rabbit mAb Coated Microwells||96 tests|
|p44/42 MAP Kinase Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Sealing Tape||2 ea|
|Cell Lysis Buffer (10X) 9803||15 ml|
The PathScan® Phospho-p44/42 MAPK (Thr202/Tyr204) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of p44/42 MAPK when dually phosphorylated at Thr202/Tyr204 of p44 (Thr185/Tyr187 of p42), and singly phosphorylated at Thr202 of p44 (Thr185 of p42). A phospho-p44/42 MAPK rabbit mAb has been coated onto the microwells. After incubation with cell lysates, phospho-p44/42 MAPK (Thr202/Tyr204) is captured by the coated antibody. Following extensive washing, a p44/42 MAPK mouse detection mAb is added to detect the captured phospho-p44/42 MAPK protein. Anti-mouse IgG, HRP-linked antibody 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 p44/42 MAPK phosphorylated at Thr202/Tyr204.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
PathScan® Phospho-p44/42 MAPK (Thr202/Tyr204) Sandwich ELISA Kit #7177 detects endogenous levels of phospho-p44/42 MAPK when phosphorylated at Thr202/Tyr204 in human and mouse cells, as shown in Figure 1. The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
Human, Mouse, Rat
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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