The Human PRAS40 [pT246] ELISA research-use-only kit is to be used for the quantitative determination of PRAS40 protein phosphorylated at threonine residue 246 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: <0.5 Units/mL • Standard curve range: 1.6–100 Units/mL • Sample type(s): cell lysate, tissue homogenate • Specificity: natural and recombinant human PRAS40 [pT246] • 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 PRAS40 [pT246] 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
Proline-Rich AKT Substrate of 40 kDa, (PRAS40; accession numbers NP_115751 and NM_032375 for the protein and mRNA sequences, respectively) is a cytosolic protein with Mr=40 kDa that was identified and described in 2003. PRAS40 appears to be expressed by all tissues, with the highest levels in liver and heart. While most proteins contain on the order of 5% proline residues, PRAS40 is unique in containing a higher percentage of proline residues, approximately 15%, localized in proline-rich regions. PRAS40’s proline-rich regions may serve as binding sites for SH3 domain and WW domain containing proteins, possibly influencing the localization or function of these proteins.
Several lines of evidence indicate that PRAS40 is a direct substrate for AKT. PRAS40 contains a consensus AKT phosphorylation site (RXRXXS/T) located at threonine 246, a sequence that is conserved in other AKT substrates, including GSK-3β, BAD, and the mammalian homolog of the C. elegans transcription factor DAF-16. Recombinant PRAS40, produced in E. coli, can be phosphorylated in vitro by purified AKT. Further evidence was obtained with an HA epitope tagged version of PRAS40, expressed transiently in HEK293 cells, that produced a phosphorylated band by western blotting using an antibody directed to the AKT substrate consensus sequence. The phosphorylation of this band was enhanced when the cells were co-transfected with a constitutively activated AKT mutant. Finally, PRAS40 phosphorylation was decreased in cells lacking AKT1 and AKT2.
Within the AKT phosphorylation site consensus sequence of PRAS40 is the consensus sequence for binding by 14-3-3 proteins (RXXpS/pT). Direct evidence for phosphorylated PRAS40’s binding to 14-3-3 proteins was revealed with in vitro binding assays, and with immunoprecipitation experiments in which c-myc tagged PRAS40 constructs were precipitated with c-myc antibody, then probed with a 14-3-3 antibody in western blotting. The 14-3-3 proteins comprise a family of evolutionarily conserved scaffolding and adaptor proteins that modulate numerous signaling events within cells by influencing protein:protein interactions, nuclear transport, and enzyme activities. These proteins play a role in regulating apoptosis by preventing the interaction of BAD and Bcl-2 following AKT activation.
Studies with wortmannin and LY294003 lend support for the existence of a phosphoinositide 3-kinase (PI3K), AKT, PRAS40, and 14-3-3-containing signaling module. Wortmannin, an inhibitor of PI3K, reduced the phosphorylation state of PRAS40 in several experimental systems. This inhibition could be prevented by expression of a constitutively activated AKT mutant. Treatment of cells with LY294003, another PI3K inhibitor, prevented PRAS40’s binding to 14-3-3 proteins.
Several stimuli enhance the level of PRAS40 phosphorylation, including insulin, NGF, and PDGF. PRAS40’s roles in conferring resistance to apoptosis, regulating cell growth, regulating glucose uptake, and in the etiology of diabetes are currently under investigation. PRAS40 is also of interest in stroke. In transient cerebral focal ischemia, a model for stroke, overexpression of phosphorylated PRAS40 was found to attenuate apoptosis.
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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