Pathway Focus: The Akt Pathway
- Accurately measure site-specific phosphorylated Akt
- More sensitive assay than western blotting
- Minimal cell lysate needed to get quantitative results
The Akt [pS473] ELISA kit (for Research Use Only) is designed to detect and quantify Akt protein phosphorylated at serine residue 473. This assay is intended for the detection of Akt [pS473] in human, mouse, or rat cell lysates using 96-well plates and a microplate reader.
Akt was initially identified as one of the downstream targets of PI 3-kinase (PI3K). Akt can be activated by a diverse array of growth factors and physiological stimuli in a PI3K-dependent manner. Activated Akt is a key mediator for cell survival, proliferation, and angiogenesis.
ELISA kits designed to measure intracellular signaling targets are 2 to 10 times more sensitive than western blotting. The improved sensitivity enables you to detect low-expressed proteins that otherwise may not be distinguishable from background. In addition, the amount of sample needed to run the assay is less than what is needed for western blots. The kit comes with a capture antibody precoated to the plate, which eliminates the need for overnight plate coating. The plates are machine-coated to help ensure low well-to-well variability. Furthermore, the plate is supplied as removable 8-well strips, allowing you to run as few samples as you want. The standards provided in the kit can be used to create a standard curve for quantitation and/or can be used as a positive control. The total assay incubation time is only 4 hours.
Learn more about Invitrogen™ ELISA Kits
|Figure 1. Jurkat cells were treated with wortmannin at various concentrations, lysed, and assayed in parallel for Akt [total] and Akt [pS473]. The level of total Akt remained comparable across the wortmannin treatment concentrations, while the level of phosphorylation at serine residue 473 decreased with increasing doses of wortmannin.|
- ABfinity™ rabbit recombinant antibodies for sensitive detection of Akt
- Enables consistent performance for your assays
- Consistent product from lot to lot
ABfinity™ recombinant rabbit antibodies are produced from specific recombinant clones to enable consistent antibody performance over time. ABfinity™ antibodies are available against many targets, including Akt pathway proteins (Akt gene ID: 11651).
Phosphorylation of threonine 308 (T308) promotes the formation of an active site in the Akt protein. This active site is necessary for Akt’s role in cell transformation and downstream signaling events. The Akt (pT308) ABfinity™ recombinant rabbbit antibody can help elucidate how potential treatments affect the up- or down-regulation of Akt activity.
Learn more about ABfinity™ Recombinant Rabbit Monoclonal Antibodies
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|Figure 2. Immunocytochemistry analysis of HeLa cells stained with AKT [pT308] ABfinity™ Recombinant Rabbit Monoclonal Antibody: (a: Green). Alexa Fluor®488 goat anti-rabbit was used as secondary, DAPI was used to stain the nucleus (b: Blue) and Alexa Fluor® 594 phalloidin for actin (c: Red). d: composite image of cells showing nuclear localization of phosphorylated AKT. e: competition with the phospho AKT [pT308] peptide.|
Product Ordering Information
|Product Name||Quantity||Cat. No.|
|AKT [pT308] ABfinity™ Recombinant Rabbit Monoclonal Antibody||100 µg||701052|
Gibco® Recombinant PDGF products offer:
- High biological activity
- High purity
- Proven compatibility with Gibco® media
Platelet-derived growth factor (PDGF) contributes to cell survival, movement, proliferation, angiogenesis, and other functions. Two classical growth factors in the PDGF family are PDGF-A and PDGF-B, active as homodimers and as the heterodimer PDGF-AB. Active growth factors bind to two receptors, PDGFR-α and PDGFR-β. Once bound, the receptors lead to activation of various pathways associated with receptor tyrosine kinase signaling, including the Akt pathway. Upon receptor activation, PI3K interacts with the receptor and activates Akt, promoting downstream events.
We offer human PDGF-AA, PDGF-AB, and PDGF-BB and mouse PDGF-BB to facilitate Akt pathway activation. In addition, we offer other growth factors to study Akt signaling.
Gibco® cytokines and growth factors are high-purity recombinant proteins with high bioactivity. Gibco® growth factors are of the highest quality because each protein is analyzed for purity along with structural homogeneity to help ensure that a biologically active protein is produced. And Gibco® cytokines and growth factors have been bioassayed with Gibco® media. Get your cytokines and growth factors from the media company you trust.
Learn more about Gibco® Cytokines and Growth Factors
|Figure 3. Immunofluorescence staining. Serum-starved NIH3T3 cells, untreated (left) or treated with PDGF (right), were fixed prior to immunostaining with the ABfinity™ Akt [pS473] rabbit monoclonal antibody. The signal was detected with a FITC-conjugated anti-rabbit secondary antibody. The results show that the antibody detected phosphorylated Akt in PDGF-treated NIH3T3 cells. Cells were counterstained with DAPI.|
- Red-fluorescent probe to detect oxidative stress
- Compatible with GFP and Alexa Fluor® 488 dye
- Suitable for live-cell imaging, enabling continuous monitoring of dynamic events
- Compatible with formaldehyde-based fixation methods, facilitating convenient multiparameter analyses
- Validated with multiple platforms, including traditional imaging, high-content screening, plate readers, and flow cytometry
Hyperactivation of Akt, a serine/threonine kinase, frequently occurs in human cancers. Recent studies indicate that Akt is involved in a fine balancing act in tumor cells: cell propagation relies on hyperactivation of Akt, but this can result in oxidative stress for the cells, and excessive reactive oxygen species (ROS) accumulation can trigger apoptosis or necrosis.
CellROX™ Deep Red reagent is a novel fluorogenic probe used to measure oxidative stress in cells. The cell-permeant dye is nonfluorescent in the reduced state but exhibits bright fluorescence upon oxidation by ROS (emission maximum ~665 nm). The signal can be monitored via fluorescence imaging, high-content imaging, fluorescence plate readers, and flow cytometers.
The bright, deep-red fluorescence signal is compatible with other live-cell dyes and GFP, making it useful in multiplex fluorescence assays for measuring a variety of cellular phenomena, including parameters related to cytotoxicity and cell death. Furthermore, unlike many other ROS sensors, the signal from CellROX™ Deep Red reagent is retained after formaldehyde fixation, to allow more flexibility in the assay and the possibility of additional downstream manipulation. In a recent publication, researchers reported that they were able to detect oxidative stress in the A-10 clonal embryonic rat aortic smooth muscle cell line using CellROX™ Deep Red reagent and fluorescence microscopy.
Learn more about CellROX™ Deep Red reagent
|Figure 4. Imaging oxidative stress with CellROX™ Deep Red reagent. Human osteosarcoma (U2OS) cells were treated with 100 μM menadione for 1 hr to induce oxidative stress, and then stained with 5 μM CellROX™ Deep Red reagent, 5 μg/mL of CellMask™ Orange plasma membrane stain, and 1 μM SYTO® Green fluorescent nuclear stain for 30 min at 37°C. The cells were washed 3 times with HBSS before imaging.|