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Figure 1. Target map of the PathScan® Intracellular Signaling Array Kit (Fluorescent Readout) #7744.

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Figure 2. MCF7 cells were grown to 80% 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® Intracellular Signaling Array Kit (Fluorescent Readout) #7744. 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 Array Vision software.

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Figure 3. HT-29 cells were grown to 80% confluency and then either untreated or UV-irradiated and allowed to recover for 60 min. Cell extracts were prepared and analyzed using the PathScan® Intracellular Signaling Array Kit (Fluorescent Readout) #7744. 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 Array Vision software.

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Figure 4. HeLa cells were grown to 90% confluency and then either untreated or treated with Staurosporine #9953 (1 μM, 3.5 hr). Cell extracts were prepared and analyzed using the PathScan® Intracellular Signaling Array Kit (Fluorescent Readout) #7744. 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 Array Vision software.

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Figure 5. NIH/3T3 cells were grown to 85% confluency and then serum starved overnight. Cells were treated with Human Platelet-Derived Growth Factor BB (hPDGF-BB) #8912 (100 ng/ml) for the indicated time periods and cell extracts were prepared and analyzed using the PathScan® Intracellular Signaling Array Kit (Fluorescent Readout). Images were acquired using the LI-COR® Biosciences Odyssey® imaging system. Pixel intensity was quantified using Array Vision software. Heatmap analysis was generated using MeV analysis software.

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PathScan® Intracellular Signaling Array Kit (Fluorescent Readout) #7744 Protocol

A. Preparing Cell Lysates

  1. Thaw 10X Cell Lysis Buffer (#9803) 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) (#8553) to a final concentration of 1 mM. Keep on ice.
  2. Remove media and wash cells once with ice-cold 1X PBS.
  3. Remove PBS and add ice-cold 1X Cell Lysis Buffer (#7018). For adherent cells, use 0.5 ml 1X Cell Lysis Buffer for each plate (10 cm in diameter). Incubate on ice for 5 minutes.
  4. If using adherent cells, dislodge the cells using a cell scraper. Transfer lysed cells to an appropriate tube. Keep on ice.
  5. 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.
  6. 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

  1. Bring glass slides and blocking buffer to room temperature before use.
  2. 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.
  3. 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.
  4. 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.

  5. Affix the multi-well gasket to the glass slide (see figure below):
    1. Place the multi-well gasket face-down on the benchtop (the silicone layer should be facing up). Remove the protective plastic film.
    2. 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.
    3. 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.
    4. 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.
    5. Snap the unmarked metal clip to the other side of the assembly in the same manner and slide into place.
    6. The assembled array is ready to use.
  6. 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.

  7. 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.
  8. 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.
  9. 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.
  10. 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.

  11. 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.
  12. Wash 4 X 5 minutes with 100 µl 1X Array Wash Buffer as in step 8.
  13. 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.
  14. 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.
  15. Remove slide from plastic dish and allow to dry completely.
  16. 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.
Slide Assembly Photos

DyLight™ is a registered trademark of Thermo Fisher Scientific Inc. and its subsidiaries.

Product Includes Quantity Cap Color
Array Slides - Intracellular Signaling Array Kit 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 Ab Cocktail (10X) - Intracellular Signaling Array Kit 300 µl White
DyLight 680TM-linked Streptavidin (10X) 300 µl Brown
PathScan® Sandwich ELISA Lysis Buffer (1X) 7018 30 ml Clear

Product Usage Information

Storage: Kit should be stored at 4°C with the exception of Lysis Buffer, which is stored at –20°C (packaged separately).

Product Description

The PathScan® Intracellular Signaling Array Kit (Fluorescent Readout) is a slide-based antibody array founded upon the sandwich immunoassay principle. The array kit allows for the simultaneous detection of 18 important and well-characterized signaling molecules when phosphorylated or cleaved. 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 576 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.


Specificity / Sensitivity

PathScan® Intracellular Signaling Array Kit (Fluorescent Readout) detects the indicated cellular proteins and signaling nodes only when phosphorylated or cleaved at the specified residues. (see Array Target Map). No significant 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

Phosphorylation and proteolysis are two widespread covalent post-translational modifications that represent important regulatory mechanisms in biology. Detection of these modifications on a set of cellular proteins playing a well-understood role in cell biology can provide a broad snapshot of intracellular signaling.

The MAPK/Erk cascade is one of the best characterized and widely studied signaling modules. It is involved in a broad range of cellular processes such as proliferation, differentiation, and motility. MAPK/Erk is activated by a wide range of extracellular signals including growth factors, cytokines, hormones, and neurotransmitters. It is activated by dual phosphorylation at Thr202 and Tyr204 by the dual specificity kinases MEK1 and MEK2.

p38 and JNK MAPKs are core components of two additional structurally related signal transduction modules. p38 and JNK are activated through a similar dual phosphorylation mechanism by various MAPK kinases in response to pro-inflammatory cytokines, stressful conditions, or genotoxicity.

Stat1 and Stat3 are important signaling molecules that are involved in immunity and inflammation and can be activated by a variety of cytokines or growth factors. Stat1 and Stat3 are phosphorylated at Tyr701 or Tyr705, respectively, by cytokine receptor-tethered tyrosine kinases of the Jak family or, in some cases, by other tyrosine kinases such as Src.

Akt is a protein kinase generally activated in response to growth factor stimulation that transmits growth and survival signals. Phosphorylation of Akt at Ser473 and Thr308 by TORC2 complex and PDK1, respectively, are reliable predictors of Akt activation. Phosphorylation of PRAS40 at Thr246 by Akt relieves PRAS40 inhibition of TORC1. Akt phosphorylation of the pro-apoptotic protein Bad at Ser112 and the multifunctional kinase GSK-3β at Ser9 inhibits their activity and promotes cell survival.

mTOR is an important signaling hub that is a major component of two macromolecular complexes, TORC1 and TORC2. mTOR is phosphorylated at Ser2448 and integrates growth factor signaling and nutrient availability, thus playing an important role in cell growth and homeostasis. mTORC1 phosphorylates p70 S6 Kinase at Thr389, leading to kinase activation and cell cycle progression. The S6 ribosomal protein is found downstream of p70 S6 Kinase and its phosphorylation at Ser235/236 reflects mTOR pathway activation and predicts cell cycle progression.

AMPK is an energy sensor that is activated by phosphorylation at Thr172 in response to elevated AMP levels. AMPK regulates fatty acid metabolism, as well as modulates protein synthesis and cell growth.

HSP27 is a mediator of cell stress that confers resistance to adverse environmental change. HSP27 is phosphorylated at Ser78 within the p38 MAPK pathway.

p53 plays an important role in cellular response to DNA damage and other genomic aberrations. Phosphorylation of p53 at Ser15 by ATM/ATR or DNA-PK in response to DNA damage leads to its stabilization and accumulation.

Caspase-3 is a critical executor of apoptosis. Caspase-3 is activated by endoproteolytic cleavage at Asp175 and exerts its pro-apoptotic activity through cleavage of multiple cellular targets. PARP, an enzyme that is involved in DNA repair, is one of the main substrates of activated caspase-3. Cleavage at Asp214 leads to PARP inactivation. Increased levels of cleaved caspase-3 and cleaved PARP are reliable indicators of apoptosis.


1.  Manning, B.D. and Cantley, L.C. (2007) Cell 129, 1261-74.

2.  Huang, J. and Manning, B.D. (2009) Biochem Soc Trans 37, 217-22.

3.  Dufner, A. and Thomas, G. (1999) Exp Cell Res 253, 100-9.

4.  Hardie, D.G. et al. (2012) Nat Rev Mol Cell Biol 13, 251-62.

5.  Rubinfeld, H. and Seger, R. (2005) Mol Biotechnol 31, 151-74.

6.  Keshet, Y. and Seger, R. (2010) Methods Mol Biol 661, 3-38.

7.  Cuadrado, A. and Nebreda, A.R. (2010) Biochem J 429, 403-17.

8.  Brognard, J. and Hunter, T. (2011) Curr Opin Genet Dev 21, 4-11.

9.  Hunter, T. (2009) Curr Opin Cell Biol 21, 140-6.

10.  Manning, G. et al. (2002) Science 298, 1912-34.

11.  Kurokawa, M. and Kornbluth, S. (2009) Cell 138, 838-54.

12.  Shi, Y. (2004) Cell 117, 855-8.

13.  Boatright, K.M. and Salvesen, G.S. (2003) Curr Opin Cell Biol 15, 725-31.


Entrez-Gene Id 207 , 208 , 10000 , 5562 , 5563 , 572 , 836 , 5595 , 5594 , 2932 , 3315 , 2475 , 1432 , 5600 , 5603 , 6300 , 7157 , 6198 , 142 , 84335 , 6194 , 5599 , 6772 , 6774


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.

7744
PathScan® Intracellular Signaling Array Kit (Fluorescent Readout)