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PathScan® Akt Signaling Antibody Array Kit (Fluorescent Readout) #9700
Figure 1. Target map of the PathScan® Akt Signaling Antibody Array Kit (Fluorescent Readout) #9700.Learn more about how we get our images
Figure 2. MCF7 cells were grown to 85% confluency and then serum starved overnight. Cells were either untreated or treated with Human Insulin-like Growth Factor I (hIGF-I) #8917 (100 ng/ml, 20 min). Cell extracts were prepared and analyzed using the PathScan® Akt Signaling Antibody Array Kit (Fluorescent Readout) #9700. Panel A shows images that were acquired using the LI-COR® Biosciences Odyssey® imaging system. Panel B shows quantification of results. Pixel intensity was quantified using the LI-COR® Image Studio v2.0 array analysis software.Learn more about how we get our images
Figure 3. A-431 cells were grown to 85% confluency and then serum starved overnight. Cells were either untreated or treated with Human Epidermal Growth Factor (hEGF) #8916 (100 ng/ml, 5 min). Cell extracts were prepared and analyzed using the PathScan® Akt Signaling Antibody Array Kit (Fluorescent Readout) #9700. Panel A shows images that were acquired using the LI-COR® Biosciences Odyssey® imaging system. Panel B shows quantification of results. Pixel intensity was quantified using the LI-COR® Image Studio v2.0 array analysis software.Learn more about how we get our images
Gallery: PathScan® Akt Signaling Antibody Array Kit (Fluorescent Readout) #9700
PathScan® Akt Antibody Array Kit (Fluorescent Readout) Protocol
A. Preparing Cell Lysates
- Thaw 10X Cell Lysis Buffer #7018 and mix thoroughly. Prepare 1X Cell Lysis Buffer by diluting 10X Cell Lysis Buffer in deionized water. Supplement 1X Cell Lysis Buffer with phenylmethylsulfonyl fluoride (PMSF) to a final concentration of 1 mM. Keep on ice.
- Remove media and wash cells once with ice-cold 1X PBS.
- Remove PBS and add ice-cold 1X Cell Lysis Buffer. For adherent cells, use 0.5 ml 1X Cell Lysis Buffer for each plate (10 cm in diameter). Incubate on ice for 5 minutes.
- If using adherent cells, dislodge the cells using a cell scraper. Transfer lysed cells to an appropriate tube. Keep on ice.
- Microcentrifuge at maximum speed for 10 minutes at 4°C and transfer the supernatant to a new tube. The supernatant is the cell lysate. Lysate may be used immediately or stored at –80°C in single-use aliquots.
- Immediately before performing the assay, dilute lysates to 0.2 – 1.0 mg/ml in Array Diluent Buffer. Set aside on ice.
B. Assay Procedure
- Bring glass slides and blocking buffer to room temperature before use.
- Prepare 1X Array Wash Buffer by diluting 20X Array Wash Buffer in deionized water. Dilute 1mL of 20X Array Wash Buffer with 19 ml of deionized water. Label as 1X Array Wash Buffer.
- Prepare 1X Detection Antibody Cocktail as follows: For running only 1 slide: Dilute 150uL of 10X Detection Antibody Cocktail with 1350 µl of Array Diluent Buffer. For running 2 slides: Dilute 300 µl of 10X Detection Antibody Cocktail with 2700 µl of Array Diluent Buffer.
- Prepare 1X DyLight 680®-linked Streptavidin as follows: For running only 1 slide: Dilute 150 µl of 10X DyLight 680®-linked Streptavidin with 1350 µl of Array Diluent Buffer. For running 2 slides: Dilute 300 µl 10X DyLight 680®-linked Streptavidin with 2700 µl of Array Diluent Buffer. *Keep on ice and protect from light.
- Affix the multi-well gasket to the glass slide (see figure at right):
- Place the multi-well gasket face-down on the benchtop (the silicone layer should be facing up). Remove the protective plastic film.
- Carefully place the glass slide on top of the multi-well gasket with the nitrocellulose pads facing down while aligning the pads with the openings in the gasket. The orientation line should appear in the upper left hand corner when the slide is oriented vertically.
- Insert the numbered metal clip into the groove in the gasket and rotate the clip into the locked position. Ensure that the clip is on the same side as the orientation line on the slide.
- Slide the clip into place. The number “1” on the metal clip will now be in the same corner of the assembly as the orientation line.
- Snap the unmarked metal clip to the other side of the assembly in the same manner and slide into place.
- The assembled array is ready to use.
Add 100 µl Array Blocking Buffer to each well and cover with sealing tape. Incubate for 15 minutes at room temperature on an orbital shaker.
Note: Do not allow the pads to dry out until after step 14.
- Decant Array Blocking Buffer by gently flicking out the liquid into a sink or other appropriate waste receptacle. Add 75 µl diluted lysate to each well and cover with sealing tape. Incubate for 2 hours at room temp (or overnight at 4°C) on an orbital shaker.
- Decant well contents by gently flicking out the liquid into a sink or other appropriate waste receptacle. Add 100 µl 1X Array Wash Buffer to each well and incubate for 5 minutes at room temperature on an orbital shaker. Repeat three more times. Decant well contents.
- Add 75 µl 1X Detection Antibody Cocktail to each well and cover with sealing tape. Incubate for 1 hour at room temperature on an orbital shaker.
Wash 4 X 5 minutes with 100 µl 1X Array Wash Buffer as in step 8.
Note: From this point on, keep slide protected from light.
- Add 75 µl 1X DyLight 680®-linked Streptavidin to each well and cover with sealing tape. Incubate for 30 minutes at room temperature on an orbital shaker.
- Wash 4 X 5 minutes with 100 µl 1X Array Wash Buffer as in step 8.
- Remove multi-well gasket by pulling the bottom of the metal clips away from the center of the slide, then peeling the slide and gasket apart.
- Place the slide face up in a plastic dish (a clean pipette tip box cover works well). Wash once for 10 seconds with 10 ml deionized water.
- Remove slide from plastic dish and allow to dry completely.
- Capture an image of the slide using a fluorescent digital imaging system capable of exciting at 680 nm and detecting at 700 nm. Quantify spot intensities using commercially available array image analysis software.
DyLight® is a registered trademark of Thermo Fisher Scientific Inc. and its subsidiaries.
|Product Includes||Quantity||Cap Color|
|Array Slides - Akt Signaling Array||2 Ea|
|16-Well Gasket||2 Ea|
|Sealing Tape||2 sheets|
|20X Array Wash Buffer||15 ml||White|
|Array Blocking Buffer||5 ml||Red|
|Array Diluent Buffer||15 ml||Blue|
|Detection Antibody Cocktail (10X)||300 µl|
|DyLight 680TM-linked Streptavidin (10X)||300 µl||Brown|
|PathScan® Sandwich ELISA Lysis Buffer (1X) 7018||30 ml||Clear|
The PathScan® Akt Signaling Antibody Array Kit (Fluorescent Readout) uses glass slides as the planar surface and is based upon the sandwich immunoassay principle. The array kit allows for the simultaneous detection of 16 phosphorylated proteins predominantly belonging to the Akt signaling network. Target-specific capture antibodies have been spotted in duplicate onto nitrocellulose-coated glass slides. Each kit contains two 16-pad slides, allowing the user to test up to 32 samples and generate 512 data points in a single experiment. Cell lysate is incubated on the slide followed by a biotinylated detection antibody cocktail. Streptavidin-conjugated DyLight® 680 is then used to visualize the bound detection antibody. A fluorescent image of the slide can then be captured with a digital imaging system and spot intensities quantified using array analysis software.
PathScan® Akt Signaling Antibody Array Kit (Fluorescent Readout) detects the indicated cellular proteins and signaling nodes only when phosphorylated on the specified residues (see Array Target Map). No substantial cross-reactivity has been observed between targets. This kit is optimized for cell lysates diluted to a total protein concentration between 0.2 and 1 mg/ml (see kit protocol).Species Reactivity: Human
The Akt signaling module is typically activated in response to growth factor stimulation of receptor tyrosine kinases transmitting primarily anabolic growth and survival signals. Akt1/2 are ubiquitously expressed protein kinases having a multitude of cellular substrates and are involved in the regulation of a wide range of cellular processes. Akt is activated by phosphorylation at two distinct sites: Ser473 by the mTORC2 complex and Thr308 by the plasma membrane residing kinase PDK1.
PI3 kinase is a lipid kinase that phosphorylates inositol phospholipids at position three to generate docking sites for Akt at the plasma membrane where Akt is activated. PTEN is a lipid phosphatase that negates the action of PI3 kinase to downregulate the signal emanating from this module.
mTOR integrates growth factor signaling and nutrient availability and is a core component of two macromolecular complexes, mTORC1 and mTORC2. The autophosphorylation of mTOR at Ser2481 correlates with the levels of its activation. mTORC1 phosphorylation of p70 S6 kinase leads to kinase activation, which in turn activates protein synthesis. The S6 ribosomal protein is found downstream of p70 S6 kinase and its phoshporylation at Ser235/236 reflects mTOR pathway activation. The mTORC2 complex activates Akt by phosphorylating it at Ser473. Phosphorylation of PRAS40 at Thr246 by Akt relieves PRAS40 inhibition of mTORC1.
4E-BP1 is a repressor of translation and inhibits cap-dependent translation initiation. Hyperphosphorylation of 4E-BP1 by mTORC1 leads to derepression of this blockade, which results in activation of cap-dependent translation.
Phosphorylation of the pro-apoptotic protein Bad at Ser112 and the multifunctional kinases GSK-3α and GSK-3β at Ser21 and Ser9, respectively, by Akt inhibits their activity and promotes cell survival.
AMPK is an energy sensor that is activated by phosphorylation at Thr172 in response to elevated AMP levels. Under conditions of low energy and elevated levels of AMP, AMPK helps to ensure that anabolic processes, such as those triggered by Akt, are decreased until energy levels are restored.
Although not a component of the Akt signaling network, Erk1 and Erk2 kinases are a central component of the Ras/MAP kinase signaling module. Erk1/2 regulate multiple cellular functions and are involved in a broad range of cellular processes, such as proliferation, differentiation, and motility. Erk and Akt signaling modules cross regulate each other at multiple points and through a variety of mechanisms. Erk is activated by a wide range of extracellular signals including growth factors, cytokines, hormones, and neurotransmitters, leading to dual phosphorylation at Thr202 and Tyr204.
The 90 kDa ribosomal S6 kinase 1 (RSK1) is activated primarily by Erk1/2 in response to many growth factors, polypeptide hormones, and neurotransmitters. p90RSK1 phosphorylates a wide range of substrates including ribosomal protein S6, and positively regulates protein translation and cellular growth. p90RSK1 can also be activated by kinases that regulate the response to cellular stress
Protein Specific References
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. PathScan is a trademark of Cell Signaling Technology, Inc. DyLight is a trademark of Thermo Fisher Scientific, Inc. and its subsidiaries. LI-COR is a registered trademark of LI-COR, Inc. Odyssey is a registered trademark of LI-COR, Inc.