Pathway Focus: The MAPK Pathway
- Easy 4 hr incubation time
- More sensitive than western blotting
- Convenient precoated, removable 8-well strips
The phosphoELISA™ kits for Research Use Only are designed to easily and reliably quantify site-specifically phosphorylated proteins or total proteins in cell lysates. A monoclonal capture antibody is coated onto the wells of the 96-well plate provided. During the first incubation, standards and unknown samples are pipetted into the wells, and the antigen of interest binds to the immobilized (capture) antibody. After washing, a rabbit antibody specific for the phosphorylation site is added to the wells. After washing, a horseradish peroxidase–labeled anti-rabbit antibody is added. This binds to the detection antibody to complete the four-member sandwich. After a third incubation and wash to remove all of the unbound HRP, a substrate solution (TMB) is added, which is acted upon by the bound HRP to produce color. The intensity of this colored product is directly proportional to the concentration of protein present, and the optical density can be read on a standard microplate reader.
There are at least three distinct mitogen-activated protein kinase (MAP kinase) signaling modules in mammalian cells that transmit extracellular signals into the nucleus to turn on the responsive genes, including ERK, JNK, and p38 kinase. The MAPK signal transduction pathway plays an essential role in regulating many cellular processes, including inflammation, cell differentiation, cell growth, and death.
ELISA kits designed to measure intracellular signaling targets are 2 to 10 times more sensitive than western blotting. The improved sensitivity enables you to detect low-expressing proteins that otherwise may not be distinguishable from background. In addition, the amount of sample needed to run the assay is less than what is needed for western blots. Each kit comes with a capture antibody precoated to the plate, which eliminates the need for overnight plate coating. The plates are machine-coated to help ensure low well-to-well variability. Furthermore, the plate is supplied with removable 8-well strips, allowing you to run as few samples as you want. The standards provided in the kit can be used to create a standard curve for quantification and/or can be used as a positive control.
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- Screened to be sensitive and specific
- Consistent performance for your assays
- Consistent product from lot to lot
ABfinity™ rabbit recombinant antibodies are produced from specific recombinant clones to enable consistent antibody performance over time. ABfinity™ antibodies are available against many targets, including MAPK pathway proteins.
The mitogen-activated protein kinase (MAPK) pathway mediates signal transduction from cell-surface receptors to transcription factors, leading 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.
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|Figure 2. Immunohistochemistry of human lung carcinoma tissue labeled with rabbit anti–ERK1/2 [pT185/pY187] . FFPE human lung carcinoma tissue was labeled with rabbit anti-ERK1/2 [pT185/pY187] (5 µg/mL). Tissue was pretreated with EDTA, then detected with the SuperPicture™ Polymer Detection Kit. Images were taken at 20x magnification. Note nuclear and cytoplasmic staining of tumor cells.|
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- Broad dynamic range—0.06 to 6,000 pmol (5 logs)
- Flexible—can be used in manual or automated, high-throughput formats
- Simple—sample dilution not necessary
Cyclic adenosine monophosphate (cAMP) is an important second-messenger in many signal transduction pathways, linking activation of cell-surface membrane receptors to intracellular responses and, ultimately, to changes in gene expression. This competitive heterogeneous immunoassay measures cAMP directly in cell lysates at levels as low as 0.06 to 6,000 pmol (60 fmol to 6 nmol), without requiring any sample dilution. Chemiluminescent detection is complete within 90 minutes and is amenable for use in automated, high-throughput screening strategies.
Among other roles and relationships between MAPK and CREB, p38 MAPK has been shown to be involved in the phosphorylation of cAMP response element binding protein (CREB) and CREB–promoter activity.
The sensitivity and simplicity of the cAMP-Screen Direct® assay enables the researcher to measure small changes in cAMP levels in investigations of cAMP and CREB’s relationship to MAPK pathways.
Learn more about cAMP-Screen® and cAMP-Screen Direct® Chemiluminescent Immunoassay Systems
Figure 3. Sensitivity and dynamic range of the cAMP-Screen Direct® system. HEK293 cells were plated at the indicated densities and grown in cAMP-Screen Direct® 96-well assay plates, followed by duplicate cAMP-Screen Direct® assays in the presence of cAMP standards. Sensitivity of the assay to exogenously added cAMP is unchanged following growth of cells in assay plates; signal intensity differences result from basal cellular levels of cAMP. Signal was measured with the TR717™ microplate luminometer.