描述：
The Human CREB [pS133] ELISA research-use-only kit is to be used for the quantitative determination of CREB protein phosphorylated at serine residue 133 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: <0.9 Units/mL
• Standard curve range: 0.8–50 Units/mL
• Sample type(s): cell lysate, tissue homogenate
• Specificity: natural and recombinant human CREB [pS133]
• 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 CREB [pS133] 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
CREB (cAMP-Response Element-Binding protein), a protein with Mr=43 kDa, is a member of the large ATF/CREM/CREB transcriptional activator family. As with other members of this family, CREB contains a highly conserved leucine zipper dimerization domain and a basic DNA binding domain at its carboxyl terminus, and a unique amino terminus. CREB is ubiquitously expressed among mammalian species, and is highly conserved evolutionarily, with numerous invertebrate, plant, and yeast homologs
CREB activates transcription in response to stimuli that elevate cytoplasmic cAMP concentrations. The series of events leading to cAMP’s activation of CREB is initiated by ligand binding to certain membrane receptors which activate adenylyl cyclase. cAMP activates a protein kinase (PKA), which translocates to the nucleus, where it phosphorylates CREB at serine 133. This phosphorylation permits CREB to recruit CREB Binding Protein (CBP), and the CREB/CBP complex in turn stimulates gene expression by interacting directly with components of the general transcriptional machinery. In addition to fostering the formation of the CREB/CBP complex, the phosphorylation of serine 133 also enhances CREB’s binding to the specific DNA sequence TGACGTCA, known as the cAMP Response Element (CRE), a sequence common to the regulatory regions of genes under the control of cAMP including Bcl-2, BDNF, the immediate early gene egr-1, and cyclin D.
In addition to stimuli that elevate cAMP levels and activate PKA, a variety of other stimuli are observed to induce CREB serine 133 phosphorylation. These include UV irradiation, cross-linking of cell membrane proteins such as surface Ig and CD28, growth factors including PDGF, NGF, EGF, FGF, and HGF, phorbol esters, serum feeding, and the Ca2+ flux that accompanies neuronal membrane depolarization. While PKA is considered to be the classical CREB kinase, other protein kinases are observed to directly phosphorylate CREB at serine 133, including the calcium/calmodulin-dependent protein kinases CaMK IV and CaMK II, Rsk -1, -2, and -3 (activated by the upstream kinase ERK1/2), and MAPKAP-K2 (activated by the upstream kinase p38).
Interestingly, phosphorylation of CREB serine 133 is found to be necessary, but not sufficient to activate transcription in many model systems. Other events required for CREB’s activation of transcription are currently being delineated. The regulation of gene expression by CREB and its role in cell growth, differentiation, and survival, as well as many areas of neuroscience, including learning and memory, regulation of mood, circadian rhythm, and drug addiction are active areas of investigation.
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.
Related links
Learn more about ELISA kits
Learn more about other immunoassays
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