The AlphaScreen® SureFire® p-Akt1/2/3 (Ser473) assay is a sandwich immunoassay for quantitative detection of phospho-Akt1/2/3 (phosphorylated on Ser473) in cellular lysates using Alpha Technology. Please note that an AlphaScreen Protein A kit, which contains the Alpha Beads necessary to run this assay, needs to be purchased separately in addition to the AlphaScreen SureFire kit.
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Alpha SureFire® kits can be used for:
In the AlphaScreen® SureFire® assay, Donor beads are coated with streptavidin to capture one of the antibodies, which is biotinylated. Acceptor beads are coated with Protein A to immobilize the other antibody. In the presence of phosphorylated protein, the two antibodies bring the Donor and Acceptor beads close together, generating signal. The amount of light emission is directly proportional to the amount of phosphoprotein present in the sample.
|Assay Target Class||Phosphoprotein|
|Experimental Type||In vitro|
|Product Brand Name||AlphaScreen SureFire|
|Second Messenger Release||Protein phosphorylation|
|Shipping Condition||Blue Ice|
|Unit Size||500 assay points|
Cellular kinase signal transduction pathways are involved in the regulation of many important cellular processes suchas 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.
Screening kinase activities in cell-based assays offers advantages over the more traditional biochemical approach of using a purified recombinant enzyme to phosphorylate a substrate, since a cell-based assay gives information on a compound’s activity in a more biological context. However, getting initial results with the AlphaScreen® SureFire® assay is highly dependent on optimal cell culture conditions, and often requires that multiple parametersbe optimized in the first set of experiments.
Akt (also known as PKB) plays a central role in cell survival andinhibiting apoptosis, by phosphorylating and inactivating several pro-apoptotic targets. Akt is activated by insulin and various growth factors, which promotes phospholipid binding and localisation at the plasma membrane, where the activation loop is phosphorylated at Thr308 by PDK1.