CST's PathScan® Total p44/42 MAPK Sandwich ELISA Kit detects endogenous levels of total p44/42 MAPK protein. Using PathScan® Phospho-p44/42 MAPK (Thr202/Tyr204) Sandwich ELISA Kit #7315, a significant induction of phospho-p44/42 MAPK can be detected in PMA-treated Jurkat cells. However, the level of total p44/42 MAPK (phospho and non-phospho), detected by this Sandwich ELISA Kit #7320, remains unchanged (Figure 1). NIH/3T3 cells treated with PMA can also be assayed using this kit. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
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.
CST's PathScan® Total p44/42 MAPK Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total p44/42 MAPK protein. A p44/42 MAPK polyclonal antibody (7315-E1*) has been coated onto the microwells. After incubation with cell lysates, both nonphospho- and phospho-p44/42 MAPK proteins are captured by the coated antibody. Following extensive washing, a p44/42 MAPK monoclonal antibody (7320-E2*) is added to detect the captured p44/42 MAPK protein. HRP-linked anti-mouse antibody #7076* is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of total p44/42 MAPK protein.
* Antibodies in the kit are custom formulations specific to the kit.
Products Volume Solution Color
p44/42 MAPK (7315-E1) Ab
Coated Microwells*96 tests
p44/42 MAPK (7320-E2) Monoclonal Detection Ab 11 ml green
Anti-mouse IgG HRP-Linked Ab 11 ml red
TMB Substrate 11 ml colorless
STOP Solution 11 ml colorless
Sealing Tape 2 sheets
20X Wash Buffer 25 ml colorless
Sample Diluent 25 ml blue
10X Cell Lysis Buffer #9803** 15 ml yellowish
*12 8-well modules -Each module is designed to break apart for 8 tests.
**Kit should be stored at 4oC with the exception of 10x Cell Lysis Buffer, which is stored at -20 oC (packaged separately).
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