The AlphaScreen® SureFire® total NFκB assay is a sandwich immunoassay for quantitative detection of NFκB (both phosphorylated and non-phosphorylated) in cellular lysates using Alpha Technology. This assay is intended to be used as a normalization for phosphorylation studies. 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 target 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 protein present in the sample.
|Assay Target Class||Protein|
|Experimental Type||In vitro|
|Product Brand Name||AlphaScreen SureFire|
|Shipping Condition||Blue Ice|
|Unit Size||500 assay points|
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.
General Information on the AlphaScreen® SureFire® Total NF-?B p65 assay,The AlphaScreen® SureFire® Total NF-?B p65 assay is used to measure endogenous NF-?B p65 in cellular lysates. The assay is an ideal system for the screening of both modulators of receptor activation (e.g. agonists and antagonists) as well as agents acting intracellularly, such as small molecule inhibitors of upstream events.
Cell lines validated for use with AlphaScreen® SureFire® kits