K-LISA mtor Activity Kit Cat. No. CBA055

User Protocol CBA055 Rev. 20 December 2006 JSW Page 1 of 8 K-LISA mtor Activity Kit Cat. No. CBA055 Note that this user protocol is not lot-specific and is representative of the current specifications for this product. Please consult the vial label and the certificate of analysis for information on specific lots. Also note that shipping conditions may differ from storage conditions. Full details are available at. Rapid, sensitive, ELISA-based mtor kinase activity assay Table of Contents Page Storage 1 Intended Use 1 Background 1 Principle of the Assay 2 Materials Provided 2 Materials Required but not Provided 3 Precautions and Recommendations 3 Reagent Preparation 3 Detailed Protocol 4 Assay Characteristics and Examples 6 References 8 Trademarks 8 Storage Upon arrival store the Glutathione-Coated 96-Well Plate, the Kinase Stop Solution, and the Anti-p70S6K-T389 at 4 C; store the mtor Standard, mtor Substrate, 100 mm DTT, and 20 mm ATP Solution at -70 C; store the remaining components of the kit at -20 C. Intended Use The K-LISA mtor 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 also useful for in vitro mtor inhibitor screening and for assessing the regulation of mtor in cell signaling. Background mtor (mammalian Target of Rapamycin) was originally identified in Saccharomyces cerevisiae where mutations of the protein kinase, TOR, confer rapamycin resistance. mtor is a large (>250 kda) class IV PI-3 kinase family member with protein kinase activity but no reported lipid kinase activity. Other members of this family include ATM, ATR, and DNA-PK. mtor forms a complex with the 12 kda cytosolic protein, FKBP-12 and rapamycin that functions to arrest the cell cycle in the G 1 phase. The G 1 block is mediated by interference with the activation of ribosomal p70s6k and cyclin dependent kinases. Cell cycle arrest by the mtor complex requires the presence of the intact kinase domain of mtor and, in particular, a conserved serine within this domain, Ser 2448, which has been identified as an Akt/PKB-mediated phosphorylation site. Phosphorylation at this site is directly related to amino acid and nutrient status. More recently it has been suggested that this nutrient effect on mtor may be carried out through AMPK, as activation of this kinase is associated with reduced signaling through mtor and reduced phosphorylation of Ser 2448. This suggests that phosphorylation of Ser 2448 might act as a switch, controlling the activity and function of mtor. Freephone 0800 100 3496 merckbiosciences.de UK Freephone 0800 622935 Ireland

User Protocol CBA055 Rev. 20 December 2006 JSW Page 2 of 8 Distinct, functional mtor complexes have been characterized in cells as being composed of either Raptor or Rictor. Both are unrelated cytosolic proteins that bind to and regulate mtor in combination with a common, third protein called GβL. Growth factor signaling, along with nutrient availability, regulates cell growth by controlling the activity of the mtor/raptor complex. The TSC complex (Tuberous Sclerosis Complex) appears to integrate these diverse signals to mtor/raptor through the small GTPase, Rheb. Energy status of the cell is relayed to TSC2 through direct phosphorylation by AMPK. mtor/raptor controls cellular growth, in part, by phosphorylating the hydrophobic motif of p70s6k and activating the kinase. mtor/raptor also phosphorylates and inhibits 4E-BPs, which inhibits the eif4e-dependent translation of capped mrnas. Recent findings indicate that mtor, when bound to Rictor instead of Raptor, activates Akt by phosphorylating its hydrophobic motif in a growth factor-sensitive manner. In this role mtor/rictor complex is proposed to be the elusive kinase, PDK2, required for full Akt activation. Biomarkers indicate that the mtor pathway is hyperactive in certain cancers, suggesting mtor as an attractive target for cancer therapy. Activated mtor may provide tumor cells with a growth advantage by promoting protein synthesis, which is the best-described physiological function of mtor signaling. mtor regulates Akt activity, a crucial downstream effector in the PI-3K PTEN pathway, which controls cell proliferation and survival. Targeting this function of mtor may have therapeutic potential. Principle of the Assay The K-LISA mtor Activity Kit is an ELISA-based activity assay that utilizes a p70s6k-gst fusion protein as a specific mtor substrate. The mtor Substrate is first bound to a solid support and mtor-containing sample is incubated with ATP in the wells of a Glutathione-Coated 96-Well Plate where active mtor phosphorylates p70s6k at Thr 389. The phosphorylated substrate is detected with Anti-p70S6K-T389 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 serves as a positive control for inhibitor analysis. Inhibition profiles can be generated based on mtor activity in the presence and absence of test inhibitor(s). The mtor Standard supplied in the kit is an enriched rat brain fraction isolated using proprietary methods. The mtor standard phosphorylates p70s6k specifically on Thr 389, and is inhibited by FKBP12/Rapamycin, a specific inhibitor of the mtor/raptor complex (Figure 3), as well as wortmannin (Figure 2), a more general PI-3K inhibitor. Materials Provided Glutathione-Coated 96-Well Plate (Kit Component No. JA9125): 1 96-well plate, pre-blocked, supplied as twelve 8-well strips mtor Substrate, (Kit Component No. JA9358): 1 vial, 55 µg, recombinant p70s6k-gst fusion protein Anti-p70S6K-T389 (Kit Component No. JA9357): 1 vial, 15 µl, specific for p70s6k phosphorylated on Thr 389 mtor Standard (Kit Component No. JA9359): 5 vials, 1 ml each, enriched rat brain fraction, sufficient for 100 tests 2X Kinase Assay Buffer (Kit Component No. JA9190): 1 bottle, 5 ml 20 mm ATP Solution (Kit Component No. KP31512): 1 vial, 1 ml 100 mm DTT (Kit Component N. KP31516): 1 vial, 1 ml Wortmannin 50X (Kit Component No. JA9189): 1 vial, 25 µl in DMSO Kinase Stop Solution (Kit Component No. KP31517): 1 bottle, 5 ml, ready-to-use 10X Plate Wash Concentrate (Kit Component No. JA9191): 1 bottle, 25 ml Antibody Diluent (Kit Component No. JA9188): 1 bottle, 25 ml, ready-to-use HRP-Antibody Conjugate (Kit Component No. JA7922): 1 vial, 100 µl, supplied as a 200x solution TMB Substrate (Kit Component No. JA1608): 1 bottle, 12 ml, ready-to-use ELISA Stop Solution (Kit Component No. JA1616): 1 bottle, 12 ml, 2.5N H 2 SO 4, ready-to-use Plate Sealer: 2 each merckbiosciences.de

User Protocol CBA055 Rev. 20 December 2006 JSW Page 3 of 8 Materials Required but not Provided Tris-buffered Saline (TBS) Wash bottle or multichannel dispenser for washing Protein assay (for cell lysates; e.g. BCA Protein Assay Kit, Cat. No. 71285 or Non-Interfering Protein Assay Kit, Cat. No. 488250) Anti-mTOR/FRAP (Ab-2) Mouse mab (22C2) (Cat. No. OP97) (optional; for immunoprecipitation of mtor from crude lysates) Protease inhibitor cocktail (e.g. Protease Inhibitor Cocktail Set III, Cat. No. 539134) Protein A or Protein G agarose for immunoprecipitation of mtor from cell lysates (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) 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. Precautions and Recommendations The divalent cation, Mn 2+, 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 Tween -20 detergent instead. mtor is sensitive to phosphate-containing buffers. PBS should not be used. The K-LISA mtor Activity Kit has been successfully used with fluorescent HRP substrates (i.e. Pierce QuantaBlu Fluorogenic Peroxidase Substrate, Cat. No.15169) as an indicator in place of the TMB Substrate supplied with the kit for colorimetric detection. The K-LISA mtor 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. Preparation of Reagents Enough reagents are supplied to process at least 100 wells. Prepare all reagents immediately prior to use. The following table 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. Reagent mtor Substrate WS Volume (per 10-well strip) 1 ml 2X Kinase Assay Buffer WS 500 µl Anti p70s6k-t389 WS HRP-Antibody Conjugate WS Plate Wash (1X) 1 ml 1 ml 20 ml Composition Dilution factor: 1:400 997.5 µl TBS 2.5 µl mtor Substrate 500 µl 2X Kinase Assay Buffer 5 µl ATP solution 10 µl 100 mm DTT Dilution factor: 1:1000 999 µl Antibody Diluent 1 µl Anti-p70S6K-T389 Dilution factor: 1:400 997.5 µl Antibody Diluent 2.5 µl HRP-Antibody Conjugate Dilution factor: 1:10 9 ml dh 2 O 1 ml 10X Plate Wash Concentrate merckbiosciences.de

User Protocol CBA055 Rev. 20 December 2006 JSW Page 4 of 8 Detailed Protocol A. 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 Standard with test inhibitor or wortmannin in a separate tube on ice for 20 min (for example 50 µl mtor Standard with 1 µl Wortmannin, 50X). This preincubation can be carried out during the incubation in step 2. 4. Aspirate contents of the wells and 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 the following components to each well in the specified order: mtor Standard or mtor Sample* (diluted to assay range with phosphate-free buffer or water) 50 µl 2X Kinase Assay Buffer WS 50 µl Total 100 µl *Pre-incubated with inhibitor in step 3 or without inhibitor 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. (Reaction may also be stopped simply by discarding the contents of the wells). 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. Wash the plate 2 more times without shaking the plate. 9. Add 100 µl Anti-p70S6K-T389 WS to each well, cover the plate with the Plate Sealer, and incubate 1 h at room temperature. 10. Wash the plate 4 times as outlined in step 8 without shaking the plate. 11. Add 100 µl HRP-Antibody Conjugate WS to each well, cover the plate with the Plate Sealer, and incubate 1 h at room temperature. 12. Wash the plate as outlined in step 10. 13. Add 100 µl TMB Substrate to each well, cover the plate with the Plate Sealer, and incubate 5-20 min at room temperature. 14. 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. merckbiosciences.de

User Protocol CBA055 Rev. 20 December 2006 JSW Page 5 of 8 B. Assay for Activity of Immunoprecipitated mtor: recommended for analysis of mtor kinase activity in samples immunoprecipitated from cell lysates. Note: K-LISA mtor Activity Kit does not include an antibody for immunoprecipitation of active mtor from cell lysates. It is important is to use an antibody that will immunoprecipitate the enzyme without affecting the catalytic 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 lysis buffer (outlined below) per 1X10 7 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. Transfer the supernatant to clean tube. 3. Pre-clear 0.5 ml cell lysate (total protein concentration = 2-5 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 o C. 4. Centrifuge at 4000 rpm for 5 min at 4 o C in a microcentrifuge to pellet the agarose beads. Transfer the precleared lysate to a fresh tube. 5. Add 5-10 µl appropriate mtor antibody (Cat. No.OP97) to the pre-cleared lysate and rotate for 1 h at 4 o 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 o C. 7. Centrifuge at 4000 rpm for 5 min at 4 o 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 (step 1 above), centrifuge at 4000 rpm for 5 min at 4 o 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. 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 o C for 30 min. 2X Kinase Assay Buffer WS 50 µl MTOR Substrate WS 50 µl Total 100 µl 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 o 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 o C. 13. Add 10-100 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. merckbiosciences.de

User Protocol CBA055 Rev. 20 December 2006 JSW Page 6 of 8 14. 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. Wash the plate 2 more times without shaking the plate. 15. Add 100 µl Anti-p70S6K-T389 WS to each well, cover the plate with the Plate Sealer, and incubate 1 h at room temperature. 16. Wash the plate 3-4 times as outlined in step 14. 17. Add 100 µl HRP Antibody-Conjugate WS to each well and incubate 1 h at room temperature. 18. Wash the plate as outlined in step 14. 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 mtor Kinase Activity 1.4 1.2 Activity (Abs 450-595) 1.0 0.8 0.6 0.4 0.2 R 2 =0.9462 0.0 0.1 1 10 100 mtor Standard (ul) Figure 1: Activity of mtor Standard. The activity of the mtor Standard (0.17-50 µl) was determined as outlined in the Detailed Protocol above. merckbiosciences.de

User Protocol CBA055 Rev. 20 December 2006 JSW Page 7 of 8 Wortmannin Inhibition of mtor Kinase Activity 100 mtor Activity (% Max) 80 60 40 20 0 0.01 0.1 1 10 100 [Wortmannin] (µm) Figure 2: Wortmannin Inhibition of mtor Activity. The activity of the mtor Standard, (50 µl) was determined as in Figure 1 in the presence of increasing concentrations of wortmannin (Cat. No. 681675; also included with the kit). Rapamycin + FKBP12 Inhibition of mtor Kinase Activity 100 mtor Activity (% Max.) 75 50 25 0 Standard Rapamycin Rapamycin + FKBP12 Wortmannin Figure 3: FKBP12 + Rapamycin inhibition of mtor Standard. The activity of the mtor Standard, (50 µl) was determined as in Figure 1 in the presence of either Rapamycin (20 µm; Cat. No. 553210 or 553211), Rapamycin (20 µm) + GST-FKBP12 (37 µg/ml), or wortmannin (10 µm; Cat. No. 681675; also included with the kit). merckbiosciences.de

mtor Kinase Activity in 293 Cell Immunoprecipitates User Protocol CBA055 Rev. 20 December 2006 JSW Page 8 of 8 1.75 1.50 mtor Activity (Abs 450-595) 1.25 1.00 0.75 0.50 0.25 0.00 background Anti mtor Control IgG Figure 4: Activity of mtor immunoprecipitated from HEK293 cell lysate. Cell lysate was prepared from HEK293 cells as described in the Detailed Protocol, section B. Cell lysates were used immediately to immunoprecipitate mtor. Equal amounts of total protein (500 µg) were immunoprecipitated with 5 µg AntimTOR/FRAP (Ab-2) Mouse mab (22C2) (Cat. No. OP97) and activity was determined as outlined in the Detailed Protocol, section B. References Chiang G.G. and Abraham R.T. 2005. J. Biol. Chem. 280, 25485. 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. Kimura, N., et al. 2003. Genes Cells 8, 65. 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. Alarcon, C.M., et al. 1996. Genes Dev. 10, 279. Brunn, G.J., et al. 1996. EMBO J. 15, 5256. Freeman, K. and Livi, G.P. 1996. Gene 172, 143. 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. Stan, R., et al. 1994. J. Biol. Chem. 269, 32027. Heitman, J., et al. 1991. Science 253, 905. Trademarks Calbiochem is a registered trademark of EMD Biosciences, Inc. Triton is a registered trademark of Dow Chemical Company. K-LISA and PhosphoSafe are trademarks of EMD Biosciences, Inc. QuantaBlu is a trademark of Pierce Biotechnology, Inc. Sold under exclusive license of allowed U.S. Patent Application 20040191836. Prices and availability are subject to change. Copyright 2005 EMD Biosciences, Inc., an affiliate of Merck KGaA, Darmstadt,. All rights reserved. Each product is sold with a limited warranty which is provided with each purchase. Each product is intended to be used for research purposes only. It is not to be used for drug or diagnostic purposes nor is it intended for human use. EMD Biosciences products may not be resold, modified for resale, or used to manufacture commercial products without written approval of EMD Biosciences. merckbiosciences.de