The Human p38 MAPK (total) ELISA research-use-only kit is to be used for the quantitative determination of p38 MAPK protein independent of its phosphorylation state in samples (see sample types indicated) using 96-well plates and a microplate reader. The assay will recognize both natural and recombinant forms of this target.
Performance characteristics
• Sensitivity: <16 pg/mL • Standard curve range: 31.2–2,000 pg/mL • Sample type(s): cell lysate, tissue homogenate • Specificity: natural and recombinant human p38 MAPK (total) • Cross-reactivity: see kit manual for cross-species and/or cross-target reactivity • Sample volume: 100 μL • Total assay time: 4 hours
Rigorous validation
Each manufactured lot of this ELISA kit is quality tested for criteria such as sensitivity, specificity, precision, and lot-to-lot consistency. See manual for more information on validation.
Principle of the method
The human p38 MAPK (total) solid-phase sandwich ELISA (enzyme-linked immunosorbent assay) is designed to measure the amount of the target bound between a matched antibody pair. A target-specific antibody has been pre-coated in the wells of the supplied microplate. Samples, standards, or controls are then added into these wells and bind to the immobilized (capture) antibody. The sandwich is formed by the addition of the second (detector) antibody, binding to the target on a different epitope from the capture antibody. A conjugated enzyme has been incorporated into the assay. After incubation and washing steps to rid the microplate of unbound substances, a substrate solution is added that reacts with the enzyme-antibody-target complex to produce measurable signal. The intensity of this signal is directly proportional to the concentration of target present in the original specimen.
Target information
p38 MAP kinase (MAPK), also known as RK (CDC2-related protein kinase) or CSBP (cytokine suppressive anti-inflammatory drug binding protein), is the mammalian homologue of the yeast HOG kinase (high osmolarity glycerol response kinase). There are at least three distinct mitogen-activated protein kinase (MAP kinase) signaling modules which mediate extracellular signals into the nucleus to turn on the responsive genes in mammalian cells, including extracellular mitogen-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK, also called stress-activated protein kinase, SAPK), and p38 kinase. The p38 signaling transduction pathway plays an essential role in regulating many cellular processes including inflammation, cell differentiation, cell growth and death.
p38 MAPK is activated in response to a variety of extracellular stimuli including osmotic shock, inflammatory cytokines, lipopolysaccharides (LPS), anisomycin, UV light, and growth factors, etc. The activation of p38 MAPK is mediated by several upstream kinases including MAP kinase-kinase 3 (MKK3), MAP kinase-kinase 6 (MKK6) and MAP kinase-kinase 4 (MKK4, also known as SEK1 and JNKK1). These kinases phosphorylate p38 at threonine 180 and tyrosine 182 in the TGY motif, resulting in p38 activation. Recently MAPKK-independent activation of p38 was shown to involve the interaction of p38α with TAB1 [transforming growth factor-beta-activated protein kinase 1 (TAK1)-binding protein 1] leading to autophosphorylation and activation of p38α. Various targets of p38 have been identified including transcription factors ATF-2, Max, MEF2C, CHOP, MAPKAPK2 (MAPK-activated protein kinase-2), and PRAK kinase (p38-related/activated protein kinase). p38 MAPK is expressed broadly in normal tissues and various cell lines. There are three alternative spliced forms of p38 (CSBP2/p38α, CSBP1, and Mxi2) as well as several homologues including p38β, p38β2, p38γ, and p38δ. These homologues are expressed at different levels in human tissues and can be activated by different, although sometimes overlapping, stress stimuli.
ELISA kits designed to measure intracellular signaling targets are typically 2–10 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.
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