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PhosphoSitePlus® Resource

  • Additional protein information
  • Analytical tools

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REACTIVITY SENSITIVITY MW (kDa) SOURCE
H
Page

A. Instrumentation

These kits were specifically designed for use with the CellSimple Cell Analyzer.

B. Kit Components

  • 10X 4-Plex Bead Cocktail
  • 10X 4-Plex Detection Antibody Cocktail
  • Streptavidin-PE Secondary
  • Array Diluent Buffer
  • 20X Array Wash Buffer
  • 1X Cell Lysis Buffer (labeled on bottle as PathScan® Sandwich ELISA Lysis Buffer)

C. Additional Items needed, but not supplied

  • 6-Tube Magnetic Separation Rack (#7017) or 12-Tube Magnetic Separation Rack (#14564)
  • Protease/Phosphotase Inhibitor Cocktail (#5872)
  • D. Preparing Cell Lysates

    Adherent cells

    1. Thaw 1X Cell Lysis Buffer #7018 and mix thoroughly. Supplement Cell Lysis Buffer with with a cocktail of protease inhibitors such as Protease/Phosphatase Inhibitor Cocktail (100X) #5872 (not included). Keep lysis buffer on ice.
    2. Culture cells under conditions of interest and desired cell culture density. Treat cells with a modulator of interest (e.g. chemical compound, growth factor, cytokine or any combination of the above) for desired time.
    3. Prior to cell lysis remove media and rinse cells once with ice-cold 1X PBS.
    4. Aspirate / remove PBS and add 0.5 mL ice-cold 1X Cell Lysis Buffer (prepared in step 1) to each plate (10 cm diameter) and incubate the plate on ice for 5 minutes.
    5. Scrape the cells off the plate and transfer to an appropriate tube. Keep on ice.
      Optional Step: Sonicate for 10-15 seconds to complete cell lysis and shear DNA (to reduce both sample viscosity and bead aggregation).
    6. Microcentrifuge for 5 min. (~18,000 rcf) at 4°C and transfer the supernatant to a new tube. The supernatant contains the cell lysate. Store at -80°C in single use aliquots.

    Suspension cells

    1. Thaw 1X Cell Lysis Buffer #7018 and mix thoroughly. Supplement Cell Lysis Buffer with with a cocktail of protease inhibitors such as Protease/Phosphatase Inhibitor Cocktail (100X) #5872 (not included). Keep lysis buffer on ice.
    2. Collect cells by low speed (~130 rcf) centrifugation for 5 minutes and remove media. Resuspend the cells with fresh medium to a cell density of 0.5-1.0 x 106 viable cells/mL. Treat cells with a modulator of interest (e.g. chemical compound, growth factor, cytokine or any combination of the above) for desired time.
    3. Collect cells by low speed centrifugation (~130 rcf) and wash once with 5-10 mL ice-cold 1X PBS.
    4. Lyse cells with 1X Cell Lysis Buffer as prepared in step 1. Roughly 2.0 mL of lysis buffer will be required to make cell extracts from 5.0 X 107 total cells (e.g. 50 mL of cell culture).
    5. Optional Step: Sonicate for 10-15 seconds to complete cell lysis and shear DNA (to reduce both sample viscosity and bead aggregation).
    6. Microcentrifuge for 5 min. (~18,000 rcf) at 4°C and transfer the supernatant to a new tube. The supernatant contains the cell lysate. Store at -80°C in single use aliquots.

    E. Assay Procedure (For Running 1 Sample)

    1. Before use, bring the 1X Cell Lysis Buffer supplemented with protease inhibitors, 10X 4-Plex Bead Cocktail, 10X 4-Plex Detection Antibody Cocktail, 10X Streptavidin-PE Secondary, Array Diluent Buffer and the 20X Array Wash Buffer to room temperature.
    2. Prepare the 1X Array Wash Buffer by diluting 20X Array Wash Buffer in deionized water. Dilute 15 mL of 20X Array Wash Buffer with 285 mL of deionized water, label and keep at room temperature.
    3. Prepare the 1X 4-Plex Detection Antibody Cocktail by diluting 21 µL of the 10X 4-Plex Detection Antibody Cocktail with 189 µL of Array Diluent Buffer. Keep the 1X Detection Antibody Cocktail on ice.
    4. Prepare the 1X Streptavidin-PE Secondary by diluting 21 µL of the 10X Streptavidin-PE Secondary with 189 µL of Array Diluent Buffer. Keep the 1X Steptavidin-PE Secondary protected from light on ice.
    5. Prepare the 1X 4-Plex Bead Cocktail solution. Vortex the 10X 4-Plex Bead Cocktail for 30 seconds. Add 20 µL of 10X 4-Plex Bead Cocktail to 180 µL of 1X Cell Lysis Buffer into a labeled microcentrifuge tube. Vortex, then magnetically separate the beads using a magnetic separation rack. Allow the beads to separate for at least 1 minute then carefully aspirate the liquid using a pipette. Remove the tube from the magnetic separation rack then add 200 µL of 1X Cell Lysis Buffer to the beads. Vortex, then place the beads aside and proceed with the sample preparation steps.
      NOTE: We recommend using either the 6-Tube Magnetic Separation Rack #7017 or the 12-Tube Magnetic Separation Rack #14654.
    6. Prepare the cell lysate sample by diluting the sample with 1X Cell Lysis Buffer to a working 1X concentration of 0.25 mg/ml.
      NOTE: The recommended final lysate protein concentration can range from 0.1 to 1.0 mg/ml.
      Optional Step: Sonicate the diluted lysate prior to adding to the washed beads to reduce aggregation.
    7. Vortex then magnetically separate the beads that were prepared as described within step 5. Carefully aspirate the liquid using a pipette.
    8. Add 200 µL cell lysate to the beads, vortex, then place on a rotator and incubate for two hours at room temperature.
    9. Spin down the microcentrifuge tube for 5 seconds using a mini-centrifuge followed by a brief vortex.
      NOTE: This step is to make sure all of the material has been spun down to the bottom of the microcentrifuge tube, followed by vortexing to break up any bead clumps that may have formed after centrifugation.
    10. Magnetically separate the beads using a magnetic separation rack for 1 minute.
    11. Carefully aspirate the liquid using a pipette.
    12. Remove the microcentrifuge tube from the magnetic separation rack then wash the beads with 200 µL of 1X Array Wash Buffer.
    13. Vortex the beads then place them back into the magnetic separation rack. Allow the beads to separate for 1 minute.
    14. Carefully aspirate the liquid using a pipette.
    15. Repeat wash steps 12 through 14 for a total of 2 washes.
    16. Add 200 µL of 1X Detection Antibody Cocktail to the beads, vortex, then place on a rotator and incubate for one hour at room temperature.
    17. Repeat wash steps 9 through 14 for a total of 2 washes.
    18. Add 200 µL of 1X Streptavidin-PE secondary to the beads, vortex, then place on a rotator and incubate for 30 minutes at room temperature protected from light.
    19. Repeat wash steps 9 through 14 for a total of 2 washes.
    20. Add 200 µL of 1X Array Wash Buffer then vortex. The sample is now ready to be analyzed on the CellSimple Cell Analyzer.
    21. Analyze 75µL samples with the CellSimple Cell Analyzer using the Multiplex Bead Assay Application. Please see the CellSimple User Guide for more details about using the Multiplex Bead Assay Application.

    posted October 2016

    revised December 2016

    Product Includes Quantity (with Count)
    10X Cell Growth Signaling 4-plex Bead Cocktail 1 x 1.2 ml
    Array Diluent Buffer 2 x 15 ml
    20X Array Wash Buffer 1 x 15 ml
    10X Cell Growth Signaling 4-Plex Detection Antibody Cocktail 1 x 1.2 ml
    10X Streptavidin-PE Secondary 1 x 1.2 ml
    PathScan® Sandwich ELISA Lysis Buffer (1X) 7018 2 x 30 ml

    Product Description

    The CellSimple™ Cell Growth Signaling Multiplex Bead Assay Kit is a bead-based 4-plex antibody assay founded upon the sandwich immunoassay principle. The assay kit allows for the simultaneous detection of four signaling molecules that are involved in the regulation of cell growth and survival. Each target-specific capture antibody has been covalently linked to a specific sized magnetic bead. Each kit contains enough reagents to run 50 samples allowing the user to generate 200 data points. The sample is incubated with the multiplex bead cocktail followed by a biotinylated detection antibody cocktail. Streptavidin linked Phycoerythrin is then used as a reporter to visualize the bound detection antibody. Analysis is then performed using the CellSimple™ Cell Analyzer, with the Multiplex Bead Assay Application.


    Product Usage Information

    Storage: All components in this kit are stable for at least 6 months when stored at the recommended temperature. Kit should be stored at 4°C with the exception of Lysis Buffer, which is stored at –20°C (packaged separately).

    Specificity / Sensitivity

    CellSimple™ Cell Growth Signaling Multiplex Bead Assay Kit detects the target proteins Phospho-PRAS40 (Thr246), Phospho-S6 Ribosomal Protein (Ser235/236), Phospho-Akt (Ser473), and Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204). No significant cross-reactivity has been observed between targets. This kit is optimized for cell lysates diluted to a total protein concentration between 0.1 and 0.5 mg/mL (see kit protocol).


    Species Reactivity: Human

    The CellSimple™ Cell Analyzer is a benchtop instrument that utilizes a disposable thin-film cassette and a combination of a 488 nm laser, two photomultiplier tubes (525/45 nm and 561 nm LP filters), Coulter Principle-based cell measurements, and on-board software to provide easy-to-run applications and data analysis. Data acquisition occurs within approximately 10 seconds per test. The instrument relies on disposable cassettes for sample handling, which alleviates the need for flow cell cleaning and fluidics maintenance and the instrument is small enough to be portable between the lab bench and the hood. Applications include quantitative assessments of cell viability, apoptosis, other labeled antibody markers and single and multiplexed bead-based assays for protein and cellular analysis.


    The fundamental cellular behaviors of growth, proliferation and survival are controlled by a number of signal transduction pathways, in particular MAPK/Erk, PI3 Kinase, and mTOR. These pathways can be activated in response to a wide range of extracellular signaling cues such as growth factors, cytokines, hormones and neurotransmitters. MAPK/Erk is activated by dual phosphorylation at Thr202 and Tyr204 by both MEK1 and MEK2 (1,2). Akt is a protein kinase generally activated downstream of the PI3 kinase signaling pathway by phosphorylation at Ser473 and Thr308, by TORC2 complex and PDK1, respectively. These phosphorylation events are reliable predictors of Akt activation (3). Phosphorylation of PRAS40 at Thr246 by Akt relieves PRAS40 inhibition of TORC1 (3). mTOR is an important signaling hub that is a major component of two macromolecular complexes, TORC1 and TORC2. mTOR integrates growth factor signaling and nutrient availability, thus playing an important role in cell growth and homeostasis (4). 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 phosphorylation at Ser235/236 reflects mTOR pathway activation and predicts cell cycle progression (5).


    1.  Di Venosa, G.M. et al. (2006) Int J Biochem Cell Biol 38, 82-91.

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

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

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

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


    Data Sheets & Documentation


    For Research Use Only. Not For Use In Diagnostic Procedures.
    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    CellSimple is a trademark of Cell Signaling Technology, Inc.

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    CellSimple™ Cell Growth Signaling Multiplex Bead Assay Kit