Activator Protein-1 (AP-1)
|Product Family||Product Number||Product Description||Technical Manual|
|IB24001-32||Human Activator Protein-1 (AP-1) Reporter Assay System, 3 x 32 assays in 96-well format||Technical Manual|
|IB24001||Human Activator Protein-1 (AP-1) Reporter Assay System, 1 x 96-well format assays||Technical Manual|
Human Activator Protein-1 Assay Kit
This Activator Protein-1 (AP-1) assay kit is an all-inclusive firefly luciferase reporter assay system that includes, In addition to AP-1 Reporter Cells, this kit provides two optimized media for use during cell culture and in diluting test samples, the reference AP-1 activator Phorbol 12-myristate 13-acetate (PMA), Luciferase Detection Reagent, and a cell culture-ready assay plate, and a detailed protocol.
AP-1 Reporter Cells are prepared using INDIGO’s proprietary CryoMite™ process. This cryo-preservation method yields high cell viability post-thaw, and provides the convenience of immediately dispensing healthy, division-competent reporter cells into assay plates. There is no need for intermediate spin-and-wash steps, viability determinations, or cell titer adjustments.
INDIGO’s AP-1 assay kits feature a luciferase detection reagent specially formulated to provide stable light emission between 5 and 90+ minutes after initiating the luciferase reaction. Incorporating a 5-minute reaction-rest period ensures that light emission profiles attain maximal stability, thereby allowing assay plates to be processed in batch. By doing so, the signal output from all sample wells, from one plate to the next, may be directly compared within an experimental set.
Kits are offered in different assay formats to accommodate researchers’ needs: 3 x 32 and 1 x 96 assay formats for screening small numbers of test compounds, as well as custom bulk reagents for HTS applications.
Bulk assay reagents can be custom manufactured to accommodate any scale of HTS. Please inquire.
Human Activator Protein-1 Assay Services
The primary application of INDIGO’s cell-based receptor assays are to quantitatively assess the bioactivity of a test compound as an agonist (activator) or antagonist (inhibition of an agonist response) of a given receptor. Service assays include a positive control reference compound and ‘vehicle’ control for every experiment. A formal study report and all data files are provided to the client upon completion of the study. To receive a quote for your proposed study, complete & submit the online “Request a Quote” form or contact an INDIGO Customer Service Representative to discuss your desired study parameters.
Human Activator Protein-1 Background
AP-1 is a dimeric transcription factor composed of homodimers or heterodimers of members of the JUN (c-Jun, JunB and JunD), FOS (c-Fos, FosB, Fra-1 and Fra-2), ATF (ATF2, ATF3,/LRF1, ATF4, ATF5, ATF6B, ATF7, B-ATF, B-ATF3, JDP1 and JDP2) and MAF (MafA, MAfB, cMAf, Nrl and MafF/G.K) families of proteins. These proteins share a characteristic basic leucine zipper (bZIP) DNA-binding domain. The leucine zipper drives the dimer formation, while the basic region is required for binding to the DNA response element. The variety of AP-1 dimer configurations confer versatility to this transcription factor in regulating numerous physiological (cell proliferation, differentiation, apoptosis, migration) and pathological activities (cancer, inflammation, transplant rejection) in the cell.
The most studied form of AP-1 transcription factor is c-Jun/Fos heterodimer. Upon activation, this heterodimer binds a palindromic DNA binding sequence referred to as the TPA response element (TRE).
AP-1 is activated by a variety of physiological and environmental stimuli such as growth factors, cytokines, stress, ultraviolet radiation, and bacterial and viral infections. The dimer composition of AP-1, the cell type and the stage of development are all important in the regulation of AP-1 activity. Upon stimulation, a cascade of kinases is activated leading ultimately to the activation of MAPK, ERK and/or JNK, which in turn regulate Fos and Jun on the transcriptional and post-translational level.
Reporter Cells contain an engineered luciferase reporter gene functionally linked to tandem TPA response element (TRE) sequences positioned immediately upstream of a minimal promoter. Activated AP-1 binds to its TRE to drive Luc gene expression. Thus, quantifying changes in luciferase activity in the treated reporter cells provides a sensitive surrogate measure of changes in AP-1 activity
Therefore, the principal application of this assay is in the screening of test samples to quantify any functional activities, either activating or inhibitory, that they may exert against AP-1.
<Synonyms: AP1, AP-1, Human Activator Protein-1, Human AP-1, Activator Protein-1
Human Activator Protein-1 Assay Data
Dose-response activation of AP-1 using PMA.
Activation of AP-1 is demonstrated by treating reporter cells with Phorbol 12-myristate 13-acetate (PMA; provided) for 22 hours, following the protocol for activation assays depicted in the assay technical manual. Average relative light units (RLU) and corresponding standard deviation (SD) values were determined for each treatment concentration (n = 4). Fold-activation and Z’ values were calculated as described by Zhang, et al. (1999). Non-linear regression and EC50 analyses were performed using GraphPad Prism software. High Z' scores confirm the robust performance of this assay.
Dose-dependent inhibition of AP-1
Human AP-1 Reporter Cells were treated with an ~EC80 concentration of PMA (10 nM) and then challenged with the inhibitors JNK Inhibitor V, CC-401, PD98059, CAY 10561, and PKC inhibitors Bisindolylmaleimide I and XI (all from Cayman Chemical, USA). Reporter cells were treated for 6 hours, following the protocol for inhibition assays depicted in the assay technical manual.