Cellomics HCS Reagent Kit for detection of activated Protein Kinase A (PKA). | | | | | PKA without
activation | treat cells | PKA
redistribution
after activation | analyze
relative to
nuclei | conclude |
DescriptionThe Cellomics PKA Activation Kit enables detection and measurement of activated cAMP-dependent protein kinase A (PKA) in stimulated cells for high-content screening. After stimulation, activated PKA redistributes from the perinuclear region (or perinuclear spots) to the cytoplasm and nucleus, producing a more diffuse staining (Figure 1). This assay measures PKA redistribution using antibody staining and is applicable across many cell types. PKA has regulatory and catalytic subunits and is inactive under low cAMP. PKA is activated through GPCR receptor signaling through the Gas protein subunit, which subsequently activates adenylate cyclase to increase cyclic AMP production. Once cAMP levels increase, the regulatory subunit binds cAMP to allow dissociation of the catalytic subunit to other cellular locations. Activated PKA phosphorylates target proteins, such as the transcription factor CREB, which regulate many cell metabolism and proliferation genes. The Cellomics PKA Activation Kits combined with the Cellomics ArrayScan HCS Reader and the Compartmental Analysis BioApplication Software Module enable quantitative monitoring of PKA activation. This kit has been optimized using NIH 3T3 cells and forskolin, an adenylate cyclase activator. NIH 3T3 cells were chosen because of the clear nuclear and cytosolic intensity differences of PKA redistribution after stimulation; however, other cell types with different PKA distributions also can be labeled with this kit and quantified by high-content screening. | Figure 1. PKA activation and redistribution in NIH 3T3 cells treated with forskolin. Cells were treated with 100 µM forskolin for 30 minutes, then fixed and stained with Hoechst 33342 dye for nuclei (blue) and PKA primary antibody plus DyLight 649 Goat Anti-Rabbit Secondary Antibody (orange). Top panels are images from non-treated cells, and bottom panels are treated cells. Columns are nuclei (blue) and PKA (orange) fluorescent channels, respectively. PKA staining is primarily perinuclear in the non-treated cells but disperses throughout the nucleus and cytoplasm upon stimulation. |
| Figure 2. Dose response curves of PKA activatin in NIH 3T3 cells treated for 30 minutes with forskolin. Each data point represents 8 wells for each point of three separate plates. Wells were scored using the output parameter of the average nuclear fluorescent intensity (MEAN_CircAvgIntCh2).
EC50 = 0.047+/-0.018 mM forskolin. |
Kit Contents| Component | 8404301 | 8404302 | | PKA Primary Antibody | 24 µl | 120 µl | DyLight 549 Conjugated
Goat Anti-Mouse IgG | 30 µl | 72 µl | | Hoechst Dye | 30 µl | 30 µl | | Wash Buffer (10X Dulbecco's PBS) | 100 ml | 100 ml | Wash Buffer II (10X Dulbecco's PBS with
Tween®-20) | 100 ml | 100 ml | Permeabilization Buffer (10X D-PBS with 1% Triton® X-100) | 100 ml | 100 ml | | Blocking Buffer (10X) | 85 ml | 85 ml | | Thin Plate Seal Assembly | 7/pack | 7/pack |
Ordering InformationScreening size kits and components (bulk and custom) available with special pricing. Please call. To order Cellomics HCS Reagent Kits, please call 800-874-3723 in the U.S.
or contact your local distributor of Thermo Scientific Products. Choose "Order Online" in table to see pricing and order online (U.S. only). See all Cellomics HCS Reagent Kits
|
Home 
Products 
top