The Alpha SureFire® Ultra™ Multiplex phospho AMPKα1/2 (Thr172) + Total AMPKα1/2 assay kit is used to measure both the phosphorylation (on Thr172) and total levels of endogenous AMPKα1/2 in cellular lysates. The assay is an ideal system for the screening of modulators of receptor activation (e.g. agonists and antagonists) as well as agents acting intracellularly, such as small molecule inhibitors of signal transduction. The assay will measure AMPKα1/2 phosphorylation by either recombinant or endogenous receptors and can be applied to primary cells.
For research use only. Not for use in diagnostic procedures.
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The Alpha SureFire Ultra Multiplex p-AMPKα1/2 (Thr172) + Total AMPKα1/2 assay kit is used to measure both phosphorylated and total levels of endogenous AMPKα1/2 in cellular lysates in a multiplexing Alpha no-wash assay. No specially engineered cell lines are required. The 615 nm (Eu) signal corresponds to the phosphorylated AMPKα1/2 analysis, and the 545 nm (Tb) signal corresponds to the total Akt1 analysis. This kit has been formulated to provide superior signal:background assay windows and to perform without interference in the presence of extraneous antibodies, making it amenable to the study of therapeutic and blocking antibodies.
In this assay, the Alpha 615 Acceptor bead is coated with the CaptSure™ antibody, which binds the CaptSure-tagged anti-phospho target antibody. The Alpha 545 Acceptor bead is coated with the CaptSure2™ agent, which binds the CaptSure2 tagged anti-total target protein antibody. The Alpha Donor bead binds the biotinylated anti-total target protein antibody.
|Assay Target Class||Phosphoprotein, Protein|
|Experimental Type||In vitro|
|Product Brand Name||AlphaLISA SureFire Ultra Multiplex|
|Shipping Condition||Blue Ice|
|Unit Size||10,000 assay points|
Evaluating pharmaceutical compound efficacy and safety in regulating cell behavior can involve the study of multiple signal transduction pathways and measuring more than one cellular target can provide greater confidence of positive compound hits on the cellular target of interest. Protein phosphorylation has been identified as a useful readout of cellular activation or inhibition, and these pathways are commonly targeted for therapeutic modulation of disease.
Numerous assay technologies are available to look at protein phosphorylation for drug development, including the AlphaLISA SureFire Ultra which provides a highly sensitive, fully homogenous option for the analysis of signaling pathways. In this application note, we describe the implementation of multiplexing with the AlphaLISA SureFire Ultra Multiplex assay, the only multiplexing platform that is truly homogenous and high throughput.
Cellular kinase signal transduction pathways are involved in the regulation of many important cellular processes such as cell survival, differentiation, and apoptosis. Kinase signaling networks are typically characterized by multiple kinases arranged in cascades containing nodes with feedback loops and crosstalk between pathways. Numerous assay technologies have been developed for studying kinase signaling pathways, and for screening compound libraries in search of agents to modify receptors or kinase activities. The quality of these assays can be impacted by data variability due to cell seeding inhomogeneity or from compound toxicity. A common method for controlling for variability is to normalize the assay signal to a cellular protein whose level does not change as a function of the treatment.
Alpha SureFire® Ultra™ Multiplex Phospho and Total assays utilize two types of Alpha Acceptor beads to simultaneously measure two signals from each assay well to easily normalize the assay. Here, we demonstrate the benefit and utility of normalizing assay signal of phosphorylated protein levels to total protein levels in two different cellular models: ERK 1/2 phosphorylation in human melanoma A375 cells and AKT 1/2/3 phosphorylation in mouse myoblast C2C12 cells.
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