Cellomics HCS Reagent Kit for detection activated protein kinase A (PKA) and phosphorylated cAMP response element binding (CREB).

Nuclei	Phospho-CREB	PKA
Measure three parameters in the same cells			Determine dose responses

Description

The Cellomics PKA and CREB Activation Kits enable simultaneous detection and measurement of activated protein kinase A (PKA) and phosphorylated cAMP response element-binding (CREB) in stimulated cells. After stimulation, PKA is activated and redistributes from the perinuclear region (or perinuclear spots) to diffuse staining in the cytoplasm and nucleus (Figure 1). Once activated, PKA phosphorylates CREB, resulting in translocation of CREB to the nucleus (Figure 1). This assay measures PKA redistribution and CREB phosphorylation using antibody staining and is applicable to many cell types.

PKA has a regulatory subunit and a catalytic subunit and is inactive under low cAMP concentrations. 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. Once activated, PKA phosphorylates target proteins, such as the transcription factor CREB, involved in the regulation of many cell metabolism and proliferation genes. PKA and CREB are classic downstream targets for Gas protein subunit in GPCR signaling.

This high-content screening (HCS) reagent kit combined with the Cellomics ArrayScan HCS Reader and the Compartmental Analysis BioApplication Software Module enable the simultaneous quantitative monitoring of PKA and CREB activation. This kit was 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 stained with this kit and quantified by HCS. CREB phosphorylation is also robust in NIH 3T3 cells and in many other cells lines.

Figure 1. Images of PKA and CREB activation after treatment with forskolin. NIH 3T3 fibroblasts were treated with forskolin (100 µM) for 30 minutes to measure redistribution of PKA and CREB phosphorylation. Cells were fixed and stained with anti-PKA and anti-phospho-CREB primary antibodies, DyLight 549 Goat Anti-Mouse, and DyLight 488 Goat Anti-Rabbit Secondary Antibodies. For nuclear staining, Hoechst 33342 Dye was used. Top panels are non-treated cells (perinuclear localization – PKA; cytosolic localization - CREB), and bottom panels are the treated cells (dispersed cytosolic localization – PKA; nuclear localization – phosphorylated CREB). Dispersion of PKA results in both cytosolic staining and nuclear staining resulting from diffuse cytosolic staining that covers the nucleus.

Figure 2. PKA and CREB activation after forskolin treatment. NIH 3T3 cells were treated with forskolin for 30 minutes and stained according to the kit protocol. Data represents mean +/-SD from three plates (eight wells per point in a plate). PKA and phospho-CREB were simultaneously measured in the same cells.

Kit Contents

Ordering Information

Screening 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).

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