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The mitogen-activated protein kinase (MAPK) pathway mediates signal transduction from cell surface receptors to downstream transcription factors that leads to cellular responses such as cell proliferation, growth, motility, survival, and apoptosis. The role of the MAPK pathway in cancer, immune disorders, and neurodegenerative diseases has been well recognized. We offer an extensive antibody portfolio to study the MAPK signaling pathway. New ABfinity™ antibodies, developed exclusively by Invitrogen, are generated by cloning immunogen-specific antibody genes and then expressing those genes in a mammalian expression system. These highly specific, high-quality monoclonal antibodies provide very reproducible results. These antibodies include phosphorylation site–specific antibodies, which are tested by phosphopeptide-blocking experiments to ensure that they only detect the phosphorylated target.
To analyze signal transduction at the protein level, researchers must study the activation and deactivation of MAPK pathway members. Most of this activation and deactivation is controlled by phosphorylation and dephosphorylation of targets.
|Using a combination of Invitrogen™ phosphorylation site–specific and total antibodies, the level of phosphorylation, and thus the level of activation and deactivation, can be determined in a samples. Each antibody lot is manufactured with patented methods under ISO standards, and tested with peptide blocking to ensure that only the highest quality, highly specific antibodies are produced. These antibodies are essential tools in studying the MAPK signaling cascade affecting cell function and diseases.|
In the typical simplified version of the MAPK pathway, there are three main events: coupling of cell surface receptors to G-proteins, a kinase cascade, and transcription/translation regulation. In an example of the first event, if EGF binds to the EGFR, then EGFR gets phosphorylated. To detect this phosphorylation event, we offer phosphorylation site–specific antibodies as well as the EGFR Sampler Pack, which includes both total and phosphorylation site–specific antibodies. Total antibodies detect EGFR before and after stimulation, but phosphorylation site–specific antibodies only produce signal after the stimulation (Figure 1), unless there is minor autostimulation in a small population of cells. For the kinase signaling cascade, we offer phosphorylation site–specific and total (pan) antibodies; Figure 2 shows immunohistochemistry performed using the ERK1/2 [pT185/pY187] ABfinity™ Recombinant Rabbit Monoclonal Antibody. And for the regulation of transcription and translation, we offer antibodies against markers such as ribosomal protein S6, RSK, and others.
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|Figure 1. Extracts of human epidermoid carcinoma (A431) cells unstimulated (-EGF) or stimulated with 200 ng/mL EGF for 15 min (+EGF) were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to nitrocellulose. The membrane was blocked with a 5% Ig-free BSA-TBST buffer overnight at 4ºC, and then incubated with each of the antibodies at 1:1,000 for 2 hr at room temperature in a 1% Ig-free BSA-TBST buffer. After washing, the membrane was incubated with goat F(ab’)2 anti–rabbit IgG alkaline phosphatase conjugate and signals were detected using the Tropix WesternStar™ method. |
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|Figure 2. Immunohistochemistry of human lung carcinoma tissue labeled with ERK1/2 [pT185/pY187] ABfinity™ Recombinant Rabbit Monoclonal Antibody. FFPE human lung carcinoma tissue was labeled with rabbit anti-ERK1/2 [pT185/pY187] (5 μg/mL). Tissues were pretreated with EDTA, then detected with SuperPicTure™ Polymer DAB. Images were taken at 20x magnification. Note nuclear and cytoplasmic staining in tumor cells.|
|Description||Reactivity, Validated (Predicted)||Applications||Quantity|| Cat. No.|
|ERK1/2 [pT185/pY187] ABfinity™ Recombinant Rabbit Monoclonal Antibody||Hu (B, Ch, Cp, Ms, Rt, X, Z)||E, IHC, WB||100 µg||700012|
|AKT [pS473] ABfinity™ Recombinant Rabbit Monoclonal Antibody||Ms, Hu (B, Ch, Cn, Cp, Eq, Fe, Ma, Mk (Rh), Rt, X, Z)||E, F, IF/ICC, IHC, WB||100 µg||700392|
|JNK1-2 [pTpY183/185] ABfinity™ Recombinant Rabbit Monoclonal Antibody (clone D12H7L17)||Hu (B, Ch, Cn, Cp, Eq, Gf, Mk (Rh), Ms, Ne, Or, Rt, Sw, X, Z)||E, F, IF/ICC, IHC, WB||100 µg||700031|
|EGFR SAMPLER PACK, Rabbit Polyclonal Antibody - UNCONJ||Hu||IHC, IP, WB||7 vials||44799G|
|RPS6 [pSpS235/6], Rabbit Polyclonal Antibody - UNCONJ||Hu (Ms, Rt)||WB||10 blots||44922G|
Reactivity: B = bovine; Ch = chicken; Cn = canine; Cp = chimpanzee; Eq = equine; Fe = feline; Gf = goldfish; Hu = human; Ma = mammalian; Mk = monkey (rhesus); Ms = mouse; Ne = nematode; Or = orangutan; Rt = rat; Sw = swine; X = Xenopus; Z = zebrafish.
Applications: E = electrophoresis; F = fluorescence; ICC = immunocytochemistry; IF = immunofluorescence; IHC = immunohistochemistry; IP = immunoprecipitation; WB = western blot.
Sensitive Kinase Detection—Measure Phosphorylation Levels of ERK, JNK, and p38 proteins using ELISA Kits
ERK (Extracellular Signal Regulated Kinase), also known as MAPK (Mitogen Activated Protein Kinase), has two closely related isoforms: ERK1 and ERK2. C-Jun N-terminal Kinase (JNK) is one of the main MAP kinase proteins in mammals. p38 MAP kinase (MAPK) is the mammalian homologue of the yeast HOG kinase (high osmolarity glycerol response kinase). These kinases belong to a family of serine/threonine kinases that are activated upon treatment of cells with a large variety of stimuli including mitogens, hormones, growth factors, cytokines, and bioactive peptides.
|Cell stimulation induces the activation of a signaling cascade, the downstream effects of which have been linked to the regulation of cell growth and differentiation as well as regulation of the cytoskeleton. The MAPK signal transduction pathway plays an essential role in regulating many cellular processes including inflammation, cell differentiation, cell growth, and death.|
Now you can investigate critical targets in MAPK signaling—ERK, JNK, and p38. Invitrogen™ ELISA kits measure phosphorylation activity and the total amount of protein in your cell lysate after inhibition or stimulation. Results are quantitative and easy to obtain. In addition, the kits are so sensitive that you can use less cell lysate sample compared to the amount needed for western blots.
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|Figure 3. ERK1/2 phosphorylation in Jurkat cells is dependent upon the levels of PMA stimulation. Jurkat cells were treated with PMA at varying concentrations (0.1 to 1,000 ng/mL) for 10 min, lysed and quantitated in parallel for ERK1/2 content (both ERK1/2 and ERK1/2 [pTpY185/187]. The amount of ERK1/2 remains constant, while the levels of phosphorylation at threonine 185 and tyrosine 187 increase with the dosage of PMA. The results correlate very well with western blot analysis of the same samples (inset).|
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|Figure 4. Recombinant p38 MAPK phosphorylated using MKK6 enzyme in vitro. Nonphosphorylated p38 MAPK was used as control. The phosphorylated and nonphosphorylated p38 MAPK were analyzed with p38 MAPK [pTpY180/182] ELISA and p38 MAPK (Total) ELISA. The p38 MAPK [pTpY180/182] ELISA kit specifically measures phosphorylated p38 MAPK at threonine 180 and tyrosine 182, and does not detect nonphosphorylated p38 MAPK protein.|
|ERK1/2 [pTpY185/187] Human ELISA Kit||Hu, Ms, Rt||96 tests||KHO0091|| |
|ERK1/2 (Total) Human ELISA Kit||Hu, Ms, Rt||96 tests||KHO0081|
|JNK1/2 [pTpY183/185] Human ELISA Kit||Hu||96 tests||KHO0131|
|JNK1/2 (Total) Human ELISA Kit||Hu||96 tests||KHO0121|
|p38 [pTpY180/182] Human ELISA Kit||Hu, Mk, Ms||96 tests||KHO0071|
|p38 (Total) Human ELISA Kit||Hu, Mk, Ms||96 tests||KHO0061|
Hu = human; Ms = mouse; Rt = rat; Mk = monkey.
|The MAPK pathway can be activated by various growth factors, including a member of the EGF-CFC family, Cripto. Cripto is upregulated in a number of cancers, making it an interesting target in cancer research. Cripto is known to bind to specific receptor tyrosine kinases through its EGF-like domain, and activates c-src and the MAPK signaling pathway, resulting in tumorigenesis and metastasis. We offer recombinant human Cripto, a bioactive recombinant protein intended for use in cell culture applications.|
|Recombinant Human Cripto||Hu||10 µg||PHC8014|| |
|Recombinant Human Cripto||Hu||25 µg||PMC8015|
|Recombinant Human Cripto||Hu||100 µg||PHC8011|
|Recombinant Human Cripto||Hu||1 mg||PHC8013|