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CBA104 K-LISA™ mTOR (Recombinant) Activity Kit

K-LISA™ mTOR (Recombinant) Activity Kit: Ficha de datos de seguridad (MSDS o SDS), certificado de análisis y de calidad (CoA y CoQ), expedientes, folletos y otros documentos disponibles.
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      Overview

      This product has been discontinued. We apologize for the inconvenience, but we do not currently have an alternative product.





      The K-LISA™ mTOR (Recombinant) Activity Kit (CBA104) is a sensitive, non-radioactive, ELISA-based assay for mTOR kinase activity. The kit is suitable for measuring the activity of purified or partially purified preparations of mTOR or mTOR that has been immunoprecipitated from crude cell lysates. The mTOR, Human, Recombinant, S. frugiperda supplied in the kit is an active fragment of mTOR (amino acids 1360-2549) purified from baculovirus-infected insect cells that can be used for in vitro screening of mTOR inhibitors.
      Catalogue NumberCBA104
      Brand Family Calbiochem®
      Materials Required but Not Delivered• Tris-buffered Saline (TBS)
      • Wash bottle or multichannel dispenser for washing
      • Spectrophotometer capable of measuring absorbance in 96-well plates at 450 nm, preferably in the dual wavelength mode at 450 nm against a reference wavelength of 540 or 595 nm.
      • Anti-mTOR/FRAP (Ab-2) Mouse mAb (22C2) (Cat. No. OP97) (optional; for immunoprecipitation of mTOR from crude lysates)
      • Protein A or Protein G agarose (e.g. Protein A Agarose Suspension, Cat. No. IP02; Protein G-PLUS Agarose Suspension, Cat. No. IP04; or Protein G-PLUS/ Protein A Agarose Suspension, Cat. No. IP05) (optional; for immunoprecipitation of mTOR from crude lysates)
      • Protease inhibitor cocktail (e.g. Protease Inhibitor Cocktail Set III, Cat. No. 539134) (optional; for immunoprecipitation of mTOR from crude lysates; see Lysis Buffer below)
      • Protein assay (for cell lysates; e.g. BCA Protein Assay Kit, Cat. No. 71285 or Non-Interfering Protein Assay™ Kit, Cat. No. 488250) (optional; for immunoprecipitation of mTOR from crude lysates)
      • Lysis Buffer (50 mM Tris HCl, pH 7.4, 100 mM NaCl, 50 mM β-glycerophosphate, 10% glycerol (w/v), 1% Tween®-20 detergent (w/v), 1 mM EDTA, 20 nM microcystin-LR, 25 mM NaF, and a cocktail of protease inhibitors (e.g. Protease Inhibitor Cocktail Set III, Cat. No. 539134) (optional; for immunoprecipitation of mTOR from crude lysates)
      References
      ReferencesChiang, G.C. and Abraham, R.T. 2007. Trends Mol. Med. 13, 433.
      Guertin, D.A. and Abraham, R.T. 2007. Cancer Cell 12, 9.
      Haar, E.V. et al. 2007. Nat. Cell Biol. 9, 316-323.
      Pearce, L.R., et al. 2007. Biochem. J. 405, 513.
      Frias, M.A., et al. 2006. Curr. Biol. 16, 1865.
      Guertin D.A., et al. 2006. Dev. Cell 11, 859.
      Jacinto, E., et al. 2006. Cell 127, 125.
      Yang, Q., et al. 2006. Genes Dev. 20, 2820.
      Ali, S.M., et al. 2005. J. Biol. Chem. 280, 19445.
      Guertin, D.A., et al. 2005. Trends Mol. Med. 11, 353.
      Sarbassov, D.D., et al. 2005. Science 307, 1098.
      Chiang, G.G. and Abraham, R.T. 2004. Methods Mol. Biol. 281, 125.
      Sarbassov, D.D., et al. 2004. Curr. Biol. 14, 1296.
      Kim, D.H., et al. 2003. Mol. Cell 11, 895.
      Kim, D.H., et al. 2002. Cell 110, 163.
      Sekulic, A., et al. 2000. Cancer Res. 60, 3504.
      Brunn, G.J., et al. 1997. Science 277, 99.
      Brunn, G.J., et al. 1996. EMBO J. 15, 5256.
      Sabers, J.S., et al. 1995. J. Biol. Chem. 270, 815.
      Zheng, X.F., et al. 1995. Cell 82, 121.
      Brown, E.J., et al. 1994. Nature 369, 756.
      Sabatini, D.M., et al. 1994. Cell 78, 35.
      Product Information
      Form96 Tests
      Format96-well plate
      Kit containsGlutathione-Coated 96-Well Plate, mTOR Substrate, mTOR Inhibitor Control, mTOR Human Recombinant S. frugiperda, Anti-p70S6K-T389, 2X Kinase Assay Buffer, 20 mM ATP Solution, 100 mM DTT, Kinase Stop Solution, 10X Plate Wash Concentrate, Antibody Diluent, HRP-Antibody Conjugate, TMB Substrate, ELISA Stop Solution, Plate Sealers, and a user protocol.
      Applications
      Biological Information
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe K-LISA™ mTOR (Recombinant) Activity Kit is suitable for measuring the kinase activity of purified or partially purified preparations of mTOR or mTOR that has been immunoprecipitated from crude cell lysates. The assay is useful for mTOR inhibitor screening or the characterization of mTOR activity.
      Storage and Shipping Information
      Ship Code Multiple Storage Conditions
      Toxicity Regulatory Review
      Hazardous Materials Attention: Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.
      Storage Multiple storage requirements
      Storage ConditionsUpon arrival store the Glutathione-Coated 96-Well Plate, the Kinase Stop Solution, the 2X Kinase Assay Buffer, and the Anti-p70S6K-pT389 at 4°C; store the mTOR Substrate, 100 mM DTT, 20 mM ATP Solution and mTOR, Human, Recombinant, S. frugiperda at -70°C; store the remaining components of the kit at -20°C.
      Note: THE KIT IS PACKAGED IN 3 BOXES. TWO BOXES ARE SHIPPED ON DRY ICE; UPON ARRIVAL, ONE OF THESE IS STORED AT -20°C AND THE OTHER IS STORED AT -70°C. THE THIRD BOX CONTAINS 2X KINASE ASSAY BUFFER, IS SHIPPED ON BLUE ICE, AND IS STORED AT 4°C; DO NOT FREEZE THE 2X KINASE ASSAY BUFFER. DO NOT FREEZE/THAW THE ANTI-p70S6K-T389 ANTIBODY. STORE RECOMBINANT mTOR AT -70°C AND AVOID MULTIPLE FREEZE/THAW CYCLES-FOLLOWING INITIAL THAW, PROMPTLY ALIQUOT ANY UNUSED PROTEIN TO PRECHILLED MICROFUGE TUBES AND STORE AT -70°C.
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsGlutathione-Coated 96-Well Plate, mTOR Substrate, mTOR Inhibitor Control, mTOR Human Recombinant S. frugiperda, Anti-p70S6K-T389, 2X Kinase Assay Buffer, 20 mM ATP Solution, 100 mM DTT, Kinase Stop Solution, 10X Plate Wash Concentrate, Antibody Diluent, HRP-Antibody Conjugate, TMB Substrate, ELISA Stop Solution, Plate Sealers, and a user protocol.
      Specifications
      Global Trade Item Number
      Número de referencia GTIN
      CBA104 0

      Documentation

      K-LISA™ mTOR (Recombinant) Activity Kit Ficha datos de seguridad (MSDS)

      Título

      Ficha técnica de seguridad del material (MSDS) 

      K-LISA™ mTOR (Recombinant) Activity Kit Certificados de análisis

      CargoNúmero de lote
      CBA104

      Referencias bibliográficas

      Visión general referencias
      Chiang, G.C. and Abraham, R.T. 2007. Trends Mol. Med. 13, 433.
      Guertin, D.A. and Abraham, R.T. 2007. Cancer Cell 12, 9.
      Haar, E.V. et al. 2007. Nat. Cell Biol. 9, 316-323.
      Pearce, L.R., et al. 2007. Biochem. J. 405, 513.
      Frias, M.A., et al. 2006. Curr. Biol. 16, 1865.
      Guertin D.A., et al. 2006. Dev. Cell 11, 859.
      Jacinto, E., et al. 2006. Cell 127, 125.
      Yang, Q., et al. 2006. Genes Dev. 20, 2820.
      Ali, S.M., et al. 2005. J. Biol. Chem. 280, 19445.
      Guertin, D.A., et al. 2005. Trends Mol. Med. 11, 353.
      Sarbassov, D.D., et al. 2005. Science 307, 1098.
      Chiang, G.G. and Abraham, R.T. 2004. Methods Mol. Biol. 281, 125.
      Sarbassov, D.D., et al. 2004. Curr. Biol. 14, 1296.
      Kim, D.H., et al. 2003. Mol. Cell 11, 895.
      Kim, D.H., et al. 2002. Cell 110, 163.
      Sekulic, A., et al. 2000. Cancer Res. 60, 3504.
      Brunn, G.J., et al. 1997. Science 277, 99.
      Brunn, G.J., et al. 1996. EMBO J. 15, 5256.
      Sabers, J.S., et al. 1995. J. Biol. Chem. 270, 815.
      Zheng, X.F., et al. 1995. Cell 82, 121.
      Brown, E.J., et al. 1994. Nature 369, 756.
      Sabatini, D.M., et al. 1994. Cell 78, 35.
      Protocolo de usuario

      Revision11-April-2013 JSW
      Form96 Tests
      Format96-well plate
      StorageUpon arrival store the Glutathione-Coated 96-Well Plate, the Kinase Stop Solution, the 2X Kinase Assay Buffer, and the Anti-p70S6K-pT389 at 4°C; store the mTOR Substrate, 100 mM DTT, 20 mM ATP Solution and mTOR, Human, Recombinant, S. frugiperda at -70°C; store the remaining components of the kit at -20°C.
      Note: THE KIT IS PACKAGED IN 3 BOXES. TWO BOXES ARE SHIPPED ON DRY ICE; UPON ARRIVAL, ONE OF THESE IS STORED AT -20°C AND THE OTHER IS STORED AT -70°C. THE THIRD BOX CONTAINS 2X KINASE ASSAY BUFFER, IS SHIPPED ON BLUE ICE, AND IS STORED AT 4°C; DO NOT FREEZE THE 2X KINASE ASSAY BUFFER. DO NOT FREEZE/THAW THE ANTI-p70S6K-T389 ANTIBODY. STORE RECOMBINANT mTOR AT -70°C AND AVOID MULTIPLE FREEZE/THAW CYCLES-FOLLOWING INITIAL THAW, PROMPTLY ALIQUOT ANY UNUSED PROTEIN TO PRECHILLED MICROFUGE TUBES AND STORE AT -70°C.
      Intended useThe K-LISA™ mTOR (Recombinant) Activity Kit is suitable for measuring the kinase activity of purified or partially purified preparations of mTOR or mTOR that has been immunoprecipitated from crude cell lysates. The assay is useful for mTOR inhibitor screening or the characterization of mTOR activity.
      BackgroundmTOR (mammalian Target of Rapamycin) is a large (~290 kDa) class IV PI-3 kinase family member. Other members of this family include ATM, ATR, and DNA-PK. mTOR regulates cell growth by controlling mRNA translation, ribosome biogenesis, metabolism, and autophagy. Distinct, functional mTOR complexes (TORCs) have been characterized in cells as being composed of mTOR and GβL/mLST8, as well as either Raptor and PRAS40 (TORC1) or Rictor, Sin1, and Protor (TORC2). The 12 kDa cytosolic protein, FKBP12, and rapamycin form a complex that inhibits TORC1. TORC1 activity is partially regulated in response to nutrient levels and is involved in the control of cellular growth, in part, by phosphorylating the hydrophobic motif and activating p70S6 Kinase (p70S6K). Phosphorylation of rapamycin-insensitive forms of p70S6K by TORC2 has been observed, and is dependent on the removal of the C-terminal domain of p70S6K. TORC1 also phosphorylates and inhibits 4E-BPs, which inhibits the eIF4E-dependent translation of capped mRNAs. Recent findings indicate that TORC2 activates Akt by phosphorylating its hydrophobic motif at Ser473 in a growth factor-sensitive manner. In this role TORC2 is proposed to be the elusive kinase, PDK2, required for full Akt activation. The mTOR pathway is dysregulated in certain cancers, suggesting mTOR as an attractive target for cancer therapy.
      Principles of the assayThe K-LISA™ mTOR (Recombinant) Activity Kit is an ELISA-based activity assay that utilizes a p70S6K-GST fusion protein as a specific mTOR substrate. The mTOR Substrate is bound to the wells of a Glutathione-Coated 96-Well Plate then incubated with recombinant mTOR-containing sample. Active mTOR phosphorylates p70S6K at Thr³⁸⁹ in the presence of ATP. The phosphorylated substrate is detected with Anti-p70S6K-pT389 antibody, followed by detection with HRP-Antibody Conjugate and TMB Substrate. Sensitivity is increased by the addition of ELISA Stop Solution, and relative activity is determined by reading the absorbance at dual wavelengths of 450/540 nm or 450/595 nm. Addition of wortmannin, a PI 3-kinase inhibitor, serves as a control for inhibitor analysis. Inhibition profiles can be generated based on mTOR activity in the presence and absence of test inhibitors.

      The mTOR, Human, Recombinant, S. frugiperda supplied in the kit is a fragment of mTOR (amino acids 1360-2549) expressed in insect cells. The mTOR kinase phosphorylates p70S6K on Thr³⁸⁹, and is inhibited by the rapamycin-FKBP12 complex, a specific inhibitor of mTOR (Figure 3), as well as wortmannin, a more general PI 3-K inhibitor (Figure 2).
      Materials provided• Glutathione-Coated 96-Well Plate (Kit Component No. JA9125-1EA): 1 plate, 96 wells, supplied as twelve 8-well strips, coated with glutathione and pre-blocked
      • mTOR Substrate (Kit Component No. JA9358-50UG): 1 vial, 50 µg, recombinant p70S6K-GST fusion protein. Refer to vial label for lot specific concentration.
      • Anti-p70S6K-pT389 (Kit Component No. JA9357-15UL): 1 vial, 15 µl, specific for p70S6K phosphorylated on Thr³⁸⁹; store at 4°C and do not freeze/thaw the antibody
      • mTOR, Human, Recombinant, S. frugiperda (Kit Component No. JA9566-10UG): 1 vial, 10 µg, recombinant mTOR fragment (amino acids 1360-2549), please see label for lot-specific concentration. Note: The specific activity will vary from lot to lot; lower amounts may be needed for lots with higher specific activity and higher amounts may be needed for lots with lower specific activity. The recommended range is 10-100 ng.
      • 2X Kinase Assay Buffer (Kit Component No. JA9574-5ML): 1 bottle, 5 ml; store at 4°C and do not freeze/thaw the buffer
      • 20 mM ATP Solution (Kit Component No. KP31512-1ML): 1 vial, 1 ml
      • 100 mM DTT (Kit Component No. KP31516-1ML): 1 vial, 1 ml
      • Wortmannin (100X) (Kit Component No. JA9565-25UL): 1 vial, 25 µl, 1 mM wortmannin in DMSO, supplied as a 100X solution
      • Kinase Stop Solution (Kit Component No. KP31517-5ML): 1 bottle, 5 ml, ready-to-use
      • 10X Plate Wash Concentrate (Kit Component No. JA9191-25ML): 1 bottle, 25 ml
      • Antibody Diluent (Kit Component No. JA9188-25ML): 1 bottle, 25 ml, ready-to-use
      • HRP-Antibody Conjugate (Kit Component No. JA7922-100UL): 1 vial, 100 µl, supplied as a 400X solution
      • TMB Substrate (Kit Component No. JA1608-12ML): 1 bottle, 12 ml, ready-to-use
      • ELISA Stop Solution (Kit Component No. JA1616-12ML): 1 bottle, 12 ml, 2.5N H₂SO₄, ready-to-use
      • Plate Sealer: 2 each
      Materials Required but not provided• Tris-buffered Saline (TBS)
      • Wash bottle or multichannel dispenser for washing
      • Spectrophotometer capable of measuring absorbance in 96-well plates at 450 nm, preferably in the dual wavelength mode at 450 nm against a reference wavelength of 540 or 595 nm.
      • Anti-mTOR/FRAP (Ab-2) Mouse mAb (22C2) (Cat. No. OP97) (optional; for immunoprecipitation of mTOR from crude lysates)
      • Protein A or Protein G agarose (e.g. Protein A Agarose Suspension, Cat. No. IP02; Protein G-PLUS Agarose Suspension, Cat. No. IP04; or Protein G-PLUS/ Protein A Agarose Suspension, Cat. No. IP05) (optional; for immunoprecipitation of mTOR from crude lysates)
      • Protease inhibitor cocktail (e.g. Protease Inhibitor Cocktail Set III, Cat. No. 539134) (optional; for immunoprecipitation of mTOR from crude lysates; see Lysis Buffer below)
      • Protein assay (for cell lysates; e.g. BCA Protein Assay Kit, Cat. No. 71285 or Non-Interfering Protein Assay™ Kit, Cat. No. 488250) (optional; for immunoprecipitation of mTOR from crude lysates)
      • Lysis Buffer (50 mM Tris HCl, pH 7.4, 100 mM NaCl, 50 mM β-glycerophosphate, 10% glycerol (w/v), 1% Tween®-20 detergent (w/v), 1 mM EDTA, 20 nM microcystin-LR, 25 mM NaF, and a cocktail of protease inhibitors (e.g. Protease Inhibitor Cocktail Set III, Cat. No. 539134) (optional; for immunoprecipitation of mTOR from crude lysates)
      Precautions and recommendations• The divalent cation, Mn2+, is required for mTOR activity.
      • mTOR is sensitive to non-ionic detergents such as Triton® X-100 and NP40. All reagents used should be free of these detergents. Cell lysis buffers should include a detergent that does not reduce the activity of mTOR and which the antibody of interest works well in. For example, Tween®-20 detergent is recommended if Anti-mTOR/FRAP (Ab-2) Mouse mAb (22C2) (Cat. No. OP97) is used as described in Detailed Protocol B.
      • mTOR is sensitive to phosphate-containing buffers. PBS should not be used.
      • The K-LISA™ mTOR (Recombinant) Activity Kit has been successfully used with mTOR immunoprecipitated from crude cell lysates (see figure 4).
      • Use ONLY polypropylene tubes for diluting and dispensing all reagents.
      • "Ultrapure" or deionized H2O (dH2O) is recommended for sample and buffer preparations.
      • It is recommended that a solvent control be included when performing inhibitor screening, as DMSO or other solvents may affect mTOR activity.
      Reagent preparationPrepare all reagents immediately prior to use. Table 1 provides reagent preparation instructions to obtain the volume of Working Solutions (WS) required for 10 wells. Volumes can be scaled to process more samples and provide overage for pipetting error.

      Dilute the mTOR Substrate according to the concentration on the vial labeled and as outlined in Table 1.

      Table 1: Preparation of Reagents

      *This will result in 100 µM ATP concentration in the final reaction. This can be altered if a lower concentration of ATP is desired for screening ATP-competitive inhibitors.**This will result in 1 mM DTT concentration in the final reaction.
      Detailed protocolA. Protocol for mTOR Kinase Activity and Inhibitor Screening: recommended for purified or partially purified mTOR preparations and for inhibitor screening.

      1. Remove the required number of strips from the Glutathione-Coated 96-Well Plate and place them in the 96-well frame. Return the unused strips to the foil pouch and reseal the entire edge of the pouch with tape. Store the remaining strips at 4°C.
      2. Add 100 µl mTOR Substrate WS to each well and incubate for 1 h at room temperature.
      3. If performing inhibitor screening/testing, pre-incubate 50 µl mTOR sample (prepared according to Table 2 with test inhibitor or Wortmannin 100X or solvent control) in a separate tube on ice for 20 min. This pre-incubation can be carried out during the incubation in step 2.

      Table 2: Preparation of mTOR sample*

      *Amount of sample needed for 1 well, prepare on ice and use prechilled components. Exception: Inhibitor WS may be prepared at room temperature.** For inhibitor screening assay; dilute inhibitor to 20X in dH2O (for example: 1 µl 100X inhibitor + 4 µl dH2O). If no inhibitor is to be included, Inhibitor WS can be substituted with 5 µl dH2O.*** Volume equivalent to 10-100 ng must be determined using lot-specific concentration listed on vial.


      4. Aspirate contents of the wells. Wash each well of the Glutathione-Coated 96-Well Plate with 200 µl TBS. Empty the contents of the wells into the sink and dry the wells by tapping the inverted plate on paper towels. Repeat for a total of 3 washes.
      5. Add 50 µl mTOR sample (prepared as described in Table 2) to each reaction well. Then add 50 µl 1X Kinase Assay Buffer WS to each reaction well (which will initiate the reaction). The final reaction volume will be 100 µl per well.
      6. Cover the plate with the Plate Sealer, mix with a plate shaker or equivalent for 30 s, and incubate for 30 min at 30°C.
      7. Stop the kinase reaction by adding 10 µl Kinase Stop Solution to each well. Proceed to step 8 within ≤20 min. (Alternatively, reaction may be stopped simply by discarding the contents of the wells and proceeding directly to step 8).
      8. Aspirate the contents of each well and wash with 200 µl Plate Wash (1X). Gently agitate the plate using a plate shaker or equivalent for 5 min. Empty the contents of the wells into the sink and dry the wells by tapping the inverted plate on paper towels.
      9. Wash the plate 2 more times without shaking the plate. For each wash step, add 200 µl Plate Wash (1X), then empty the contents of the wells into the sink and dry the wells by tapping the inverted plate on paper towels.
      10. Add 100 µl Anti-p70S6K-pT389 WS to each well, cover the plate with the Plate Sealer, and incubate 1 h at room temperature.
      11. Wash the plate 4 times as outlined in step 9 without shaking the plate.
      12. Add 100 µl HRP-Antibody Conjugate WS to each well, cover the plate with the Plate Sealer, and incubate 1 h at room temperature.
      13. Wash the plate 4 times as outlined in step 9 without shaking the plate.
      14. Add 100 µl TMB Substrate to each well, cover the plate with the Plate Sealer, and incubate 5-20 min at room temperature.
      15. Add 100 µl ELISA Stop Solution to each well and read the absorbance at 450 nm, preferably with a reference wavelength set at 540 or 595 nm.

      B. Assay for Activity of Immunoprecipitated mTOR: recommended for analysis of mTOR kinase activity in samples immunoprecipitated from cell lysates. Note: K-LISA™ mTOR (Recombinant) Activity Kit does not include lysis buffer or an antibody for immunoprecipitation of active mTOR from cell lysates. It is important to use an antibody that will immunoprecipitate the enzyme without affecting the activity of the kinase and can efficiently immunoprecipitate mTOR from crude biological samples.
      1. Wash cells 2 times with TBS and prepare cell lysates by adding 1 ml of prechilled Lysis Buffer (outlined below) per 1 X 107 cells. Incubate on ice for 20 min.

      Lysis buffer: 50 mM Tris HCl, pH 7.4, 100 mM NaCl, 50 mM β-glycerophosphate, 10% glycerol (w/v), 1% Tween®-20 detergent (w/v), 1 mM EDTA, 20 nM microcystin-LR, 25 mM NaF, and a cocktail of protease inhibitors (e.g. Protease Inhibitor Cocktail Set III, Cat. No. 539134).

      2. Pellet the insoluble material by centrifuging the lysate at 20,000 x g for 10 min at 4°C. Transfer the supernatant to a clean tube.
      3. Pre-clear 0.5 ml cell lysate (total protein concentration = 1-5 mg/ml) by adding 15 µl Protein G-Plus, Protein A, or Protein A/Protein G-Plus agarose beads (settled bed volume) and incubating for 15 min at 4°C.
      4. Centrifuge at 4000 rpm for 5 min at 4°C in a microcentrifuge to pellet the agarose beads. Transfer the pre-cleared lysate to a clean tube.
      5. Add appropriate mTOR antibody (e.g. 5 µg Anti-mTOR/FRAP (Ab-2) Mouse mAb (22C2) Cat. No.OP97) to the pre-cleared lysate and rotate for 1 h at 4°C.
      6. Add 50 µl Protein G-Plus, Protein A, or Protein A/Protein G-Plus agarose beads (settled bed volume) and rotate 60-90 min at 4°C.
      7. Centrifuge at 4000 rpm for 5 min at 4°C in a microcentrifuge to pellet the agarose beads (now bound to immunoprecipitated mTOR protein). Carefully remove the supernatant with a pipet tip and discard.
      8. Wash the agarose beads with 0.5 ml Lysis Buffer, centrifuge at 4000 rpm for 5 min at 4°C to pellet the agarose beads, and discard the supernatant. Repeat for a total of 3 washes. Wash the pellet one time in 1X Kinase Assay Buffer WS. Carefully remove excess liquid following the final wash so as not to disturb the loose pellet of agarose beads.
      9. Add the following components in the specified order to the agarose beads in each tube, gently mix to evenly suspend the beads, and incubate at 30°C for 30 min.

      Table 3: Assay Setup for Activity of Immunoprecipitated mTOR


      10. Add 10 µl Kinase Stop Solution to each tube and mix briefly by gently tapping the tube. DO NOT VORTEX.
      11. Centrifuge at 4000 rpm for 5 min at 4°C to pellet the agarose beads. Transfer supernatant to a fresh tube. The supernatant should be used immediately or stored at -70°C until the assay can be completed.
      12. Remove required number of strips from the Glutathione-Coated 96-Well Plate and place them in a 96-well frame. Return the unused strips to the foil pouch and seal the entire edge of the pouch with tape. Store the remaining strips at 4°C.
      13. Add 50-100 µl supernatant from step 11 to designated wells. (If less than 50 µl is used, add 1X TBS to a final volume of 50 µl.) Incubate for 30-60 min at 30°C.
      14. Aspirate the contents of each well and wash with 200 µl Plate Wash (1X) WS. Gently agitate the plate using a plate shaker or equivalent for 5 min. Empty the contents of the wells into the sink and dry the wells by tapping the inverted plate on paper towels. Wash the plate 2 more times without shaking the plate.
      15. Add 100 µl Anti-p70S6K-pT389 WS to each well, cover the plate with the Plate Sealer, and incubate 1 h at room temperature.
      16. Wash the plate 4 times as outlined in step 14 (without shaking the plate).
      17. Add 100 µl HRP Antibody-Conjugate WS to each well and incubate 1 h at room temperature.
      18. Wash the plate 4 times as outlined in step 14 (without shaking the plate).
      19. Add 100 µl TMB Substrate to each well and incubate 5-20 min. at room temperature.
      20. Add 100 µl ELISA Stop Solution to each well and read the absorbance at 450 nm, preferably with a reference wavelength set at 540-595 nm.
      Assay characteristics and examples

      Figure 1: Activity of Recombinant mTOR

      The activity of increasing amounts of mTOR, Human, Recombinant, S. frugiperda (1-200 ng) was determined as outlined in Detailed Protocol A.


      Figure 2: Inhibition of mTOR Activity by Wortmannin

      The activity of mTOR, Human, Recombinant, S. frugiperda was determined as outlined in Detailed Protocol A in the presence of increasing concentrations of wortmannin (Cat. No. 681675).


      Figure 3: Inhibition of mTOR Activity by FKBP12 + Rapamycin

      The activity of mTOR, Human, Recombinant, S. frugiperda was determined as described in Detailed Protocol A, in the presence or absence of 3 µg/ml FKBP12 (Cat. No. 325902) and increasing concentrations of Rapamycin (Cat. No. 553210 or 553211). Inhibitor WS was prepared with 1 µl of 100X rapamycin, 1 µl of 100X (300 µg/ml) FKBP12, and 3 µl dH2O and incubated on ice for 20 min; mTOR was added and the mixture was incubated 20 min on ice prior to step 5.


      Figure 4: Activity of mTOR Immunoprecipitated from HEK293 Cell Lysate

      Cell lysate was prepared from HEK293 cells as described in the Detailed Protocol B. Cell lysates were used immediately to immunoprecipitate mTOR. Equal amounts of total protein (500 µg) were immunoprecipitated with 5 µg Anti-mTOR/FRAP (Ab-2) Mouse mAb (22C2) (Cat. No. OP97) or 5 µg normal mouse IgG and activity was determined as outlined in Detailed Protocol B.
      Registered TrademarksCalbiochem® is a registered trademark of Merck KGaA, Darmstadt
      Tween® is a registered trademark of ICI Americas, Inc.
      Triton® is a registered trademark of Dow Chemical Company.
      K-LISA™ and Interactive Pathways™ are trademarks of Merck, KGaA, Darmstadt.