MEK1/2 (MAPK Kinase) Activity Assay Kit

MEK1/2 (MAPK Kinase) Activity Assay Kit For 96 tests Cat. No. SGT440 FOR RESEARCH USE ONLY Not for use in diagnostic procedures USA & Canada Phone: +1(800) 437-7500 Fax: +1 (951) 676-9209 Europe +44 (0) 23 8026 2233 Australia +61 3 9839 2000 Germany +49-6192-207300 ISO Registered Worldwide www.chemicon.com custserv@chemicon.com techserv@chemicon.com

Application Mammalian cells respond to external stimuli by activation of a variety of signal transduction pathways which result in proliferation, hypertrophy, differentiation or apoptosis. Upon growth factor or cytokine treatment, transmission of stimulatory signals from receptors to the nuclear targets appears to involve the regulation of the activity of a family of kinases known as MAPKs (mitogenactivated protein kinases) or ERKs (extracellular signal regulated kinases) (Figure 1)[1,2]. For example, the ability of growth factors to promote proliferation depends on the activation of receptor tyrosine kinases, which recruit the Ras family small G proteins and lead to the sequential activation of Raf (MAPK kinase kinase), MEK (MAPK kinase), and ERK (MAPK) [1-4]. MAPK/ERK activity requires phosphorylation on both threonine (T185) and tyrosine (Y187) [3]. The MAP kinase cascade is an evolutionarily conserved signaling pathway that plays a central role in integrating the signals from a diverse group of extracellular stimuli and proto-oncogenes to the nucleus where activation of specific transcription factors elicits cellular responses including proliferation, differentiation, survival, and apoptosis in all eukaryotes [5, 6]. Figure 1 1

CHEMICON s non-radioactive MEK1/2 Activity Assay provides a simple, convenient, and specific method for measuring MEK1/2 activity. Testing of purified MEK enzyme, in vitro inhibitor screening and the study of MEK regulation can be performed with this assay. Test Principle CHEMICON s non-radioactive MEK 1/2 Activity Assay Kit includes recombinant inactive ERK2 protein, Biotinylated MBP Substrate, a purified Phospho-MBP specific monoclonal antibody, a secondary antibody conjugated to horseradish peroxidase (HRP) and other components required to perform 96 ELISA-based assays. The Biotinylated MBP Substrate contains multiple phosphorylation sites and can be phosphorylated by a wide range of kinases. To test for the presence of MEK activity the sample is mixed with inactive ERK2 and the MBP substrate. If active MEK is present, it will phosphorylate ERK2, which in turn will phosphorylate the Thr 97 residue on the Biotinylated MBP Substrate. After quenching the enzyme reaction with an inhibitor, both the phosphorylated and dephosphorylated substrates are captured on the Streptavidin-Coated Plate. The fraction of phosphorylated substrate is visualized using a phospho-mbp specific monoclonal antibody and an HRP conjugated secondary antibody, followed by a chromogenic substrate reaction (Figure 2). Reagents and materials supplied in the MEK 1/2 Activity Assay Kit are sufficient for 96 reactions. Figure 2: Test Principle The CHEMICON MEK 1/2 Activity Assay Kit is intended for research use only; not for diagnostic or therapeutic applications. 2

Kit Components 1. ERK2, Recombinant Human (Inactive) (Part No. 90351): One vial containing 50 µg of human recombinant GST protein at 0.5 mg/ml. Store at 20 C. Use 1 µl per 50 µl reaction. 2. Streptavidin-Coated Plate (Part No. 90190): One 96-well plate with 8 X 12 well strips. 3. MAP Kinase Substrate (Part No. 90213): One vial containing 100 µg of biotinylated MBP at 1.0 mg/ml. Dilute 1:5 in 5X Kinase Assay Buffer, use 2 µl per 50 µl reaction. Store at -20 C. 4. Mouse Anti Phospho-MBP Antibody (Part No. 90212): One 100 µl vial. Dilute 1:100 in Blocking Solution before use. 5. 5X Kinase Assay Buffer (Part No. 90210): One 12 ml bottle (5X) containing 250 mm Tris, ph 7.5, 50 mm MgCl 2, 0.1% BSA. Use 10 µl per 50 µl reaction, add DTT at a final concentration of 1-5 mm before use. 6. 5X ATP/MgCl 2 Solution (Part No. 90194): One 1.5 ml vial (5X) containing 5 mm ATP and 50 mm MgCl 2 in TBS (25 mm Tris, 0.15 M NaCl) ph 7.2, with 0.01% thimerosal added as preservative. Use 10 µl per 50 µl reaction. 7. HRP-conjugated Secondary Antibody (Part No. 90211): One lyophilized vial. Dilute 1:5000 in PBS before use. 8. Blocking Solution (Part No. 90195): One 50 ml bottle (Ready to Use). 9. Wash Buffer, 10X (Part No. 60245): One 100 ml bottle of a 10X solution. Dilute to 1X with distilled water. 10. Substrate Solution (Part No: 90028): One 12 ml bottle of 3,3,5,5 - tetramethylbenzidine (TMB) (Ready to Use). 11. Stop Solution (Part No: 60193): One 12 ml bottle (Ready to Use). 3

Materials Not Supplied Reagents: MAP Kinase Inhibitor - Kinase inhibitors are required for stopping the kinase reaction after incubation with the MBP substrate. Several kinase inhibitors are available, for example EDTA. Lysis Buffer - such as RIPA buffer; 50 mm Tris ph 8.0, 150 mm NaCl, 0.5 mm EDTA, 1 mm DTT, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS, 100 µg/ml PMSF*, 1 µg/ml Aprotinin*, 2 µg/ml Leupeptin*, 100 µm Sodium Vanadate. Note: Protease Inhibitors (PMSF*, Aprotinin*, Leupeptin*) should be added freshly from stock solution directly before each use. DTT Distilled water (for dilution of the 10X Wash Buffer) PBS Equipment: 96-well Microplate Reader, 450nm 37 C Water Bath or Incubator 30 C Water Bath or Incubator Optional: Anti-MEK antibody for Immunoprecipitation. Protein A and/or G Beads (for immunoprecipitation) MEK1-GST Fusion Protein as a positive control (Chemicon Cat. No. SGT220). 4

Preparation of Reagents 1. MAP Kinase Substrate: Immediately before use dilute the MBP stock 1:5 with 1X Kinase Assay Buffer. Do not store diluted MBP solutions. 2. 5X Kinase Assay Buffer: Immediately before use add DTT to the 5X Kinase Assay Buffer (final concentration of 1-5 mm). 3. Mouse Anti Phospho-MBP Antibody: Immediately before use dilute the antibody 1:100 with Blocking Buffer. Do not store diluted solutions. 4. HRP-conjugated Secondary Antibody: Reconstitute the secondary antibody with 100 µl of 50% Glycerol. Aliquot and store the reconstituted antibody solution at 20 C. Immediately before use dilute the HRP conjugated secondary antibody solution 1:5000 with 1X PBS. Do not store diluted solutions. 5. Wash Buffer (10X): Dilute wash buffer 1:10 with distilled water for a 1X Wash Buffer. Sample Preparation Various extraction and purification methods can be utilized to isolate MAPK Kinases from blood, solid tissue or cell culture. The following protocols are provided as EXAMPLES of suitable methods. It is strongly advised that an initial experiment should be performed to determine the proper dilution of the crude sample or purified MAPK Kinase to be used in subsequent experiments. Solid Tissue Extracts: 1. Homogenize fresh tissue (brain, liver, etc.) one to four volumes of a prechilled detergent lysis buffer (RIPA buffer). 2. Centrifuge the homogenate at 12,000 x g for 10 minutes at 4 C to pellet the insoluble fraction. 3. Remove clear supernatant carefully from pellet. This supernatant is your total protein extract. It should be stored at 70 C. Note: Every freeze-thaw cycle will decrease the enzymatic activity of protein extract. 5

Cell Culture Lysates: 1. Remove cell media and wash once with pre-chilled PBS. 2. Adherent cells: Add 1 ml/100 mm dish of the pre-chilled detergent lysis buffer. Place culture dish on ice for 10 minutes. Harvest cells with rubber policeman and collect cell lysate by centrifugation at 12,000 x g for 10 minutes. Suspension cells: Place cells on ice. Add 1 ml/10 7 cells of pre-chilled detergent lysis buffer and lyse resuspended cells using either a homogenizer, sonication, or three cycles of freezing and thawing. 3. If necessary, to further clarify lysate, transfer the extracts to microcentrifuge tubes and centrifuge at 12,000 x g for 10 minutes. 4. Collect clear lysate and store at 70 C. This supernatant is your cytosolic fraction. Immunoprecipitation: 1. Add 10-20 µl of anti-mek antibody to 0.5-1 ml of cell lysate. Incubate 1-12 hours at 4 C on a shaking or rocking platform. 2. Add 50-100 µl of protein A and/or G agarose beads (50% slurry) and incubate for 1-4 hours at 4 C on a rocking/shaking platform. 3. Collect protein A/G - antigen - antibody complexes by centrifugation at 2000 x g for 1 minute at 4 C. 4. To wash, remove supernatant carefully and add 1 ml cell lysis buffer to the beads, resuspend and incubate for 10 minutes at 4 C on a rocking/shaking platform. 5. Collect protein A/G antigen - antibody complexes by centrifugation at 2000 x g for 1 minute at 4 C. 6. Repeat steps 4 and 5 once more. 7. Repeat steps 4 and 5 once more, using the kinase assay buffer instead of cell lysis buffer. 8. Remove supernatant carefully. The precipitated protein bound to the beads (IP beads) can be used directly for the kinase assay (see Assay Protocol). 6

Assay Protocol Table 1: Composition of reaction mix, total volume should be 50 µl. Sample MEK Sample IP Beads ERK (unactive) 5X Kinase Assay Buffer 5X ATP/MgCl2 Solution Diluted Kinase Substrate H2O Inhibitor Negative Control MEK Sample - - 1µL 10µL 10µL 2µL 1-25µL - 1µL 10µL 10µL 2µL MEK IP - 1-25µL 1µL 10µL 10µL 2µL MEK IP Negative Control Kinase Inhibitor - 1-25µL - 10µL 10µL 2µL 1-25µL - 1µL 10µL 10µL 2µL QS to 50µL QS to 50µL QS to 50µL QS to 50µL QS to 50µL - - - - 10µL 1. Prepare the reaction mixture according to the table above and start the reaction by adding 10 µl of 5X ATP/MgCl 2 solution. 2. Incubate the reaction at 30 C. The phosphorylation reaction time will vary from a few minutes to 60 minutes (determined by end user), a recommended starting reaction time is 30 minutes. 3. Terminate enzyme reaction by adding 10 µl of kinase inhibitor, such as 120 mm EDTA. 4. Transfer the reaction mixture to a Streptavidin-Coated strip and incubate at 37 C for 30 minutes. (Note: For IP samples, briefly centrifuge the beads adding the supernatant to the well.) 5. Aspirate the sample and wash the plate four times with 250 µl 1X Wash Buffer. A thorough washing of the plate is important to reduce background. Aspirate the wash buffer with a vacuum manifold or by inverting the plate over a sink and blotting the plate onto an absorbent surface. 6. Add 200 µl of Blocking Solution to each well and incubate at 37 C for 30 minutes. 7

7. Aspirate the Blocking Solution and add 100 µl of diluted Mouse Anti Phospho-MBP antibody to each reaction well. Incubate for 1 hour at room temperature on a shaking platform. 8. Aspirate the antibody and wash the plate four times with 250 µl of 1X Wash Buffer as described in step 5. 9. Add 100 µl of the diluted HRP conjugated secondary antibody solution to each reaction well. Incubate for 30 minutes at room temperature. 10. Aspirate the antibody and wash the plate four times with 250 µl of 1X Wash Buffer as described in step 5. 11. Add 100 µl of Substrate Solution to each well. Incubate at room temperature for 5 15 minutes (time determined by end user). 12. Stop the enzyme reaction by adding 100 µl of Stop Solution to each well. 13. Read the plate (< 5 minutes) to determine absorbance values using a spectrophotometric plate reader set at λ 450. Absorbance values will decrease over time. Note: Background reading of Negative Control should be subtracted from the readings of the kinase reaction samples before calculating the MEK Kinase activity. Storage Store the recombinant ERK2 (inactive) protein and MAP Kinase Substrate at -20 C up to their expiration date. Store all other kit components at 2 to 8 C up to their expiration date. 8

Calculation of Results Relative optical density (OD) values obtained with the CHEMICON MEK1/2 Activity Assay Kit may be compared with known standards or other test samples to obtain relative activities. Therefore, if required, purified MEK1 or MEK2 can be used for the protein kinase activity curve. Figure 3 illustrates typical results upon using purified MEK1. Kinase reaction time is 30 minutes and incubation time of TMB substrate is 5 minutes. It is always recommended to run a negative control sample. The data below is for reference only, this data should not be used to interpret actual assay results. Figure 3: Example of MEK Activity Assay Purified active MEK1 (Cat. No. SGT220), inactive ERK 2 (Cat. No. 90351), and biotinylated MBP substrate were incubated for 30 minutes at 30 C and activity was detected as described in the Assay Protocol. MEK 1/2 Kinase Activity Assay 1.5 1.2 OD 450nm 0.9 0.6 0.3 0 0 100 200 300 400 500 MEK1 [ng/rxn] 9

References 1. Cano E and Mahadevan LC. (1995) Trends Biochem Sci. 20, 117-122. 2. Lewis TS, Shapiro PS, and Ahn NG. (1998) Advances in Cancer Research. 74, 49-139. 3. Leevers SJ and Marshall CJ. (1992) EMBO. 11, 569-574. 4. Sozeri O et al. (1992) Oncogene. 7, 2259-2262. 5. Blenis, J. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 5889-5892. 6. Cobb, MH et al. (1994) Seminars in Cancer Biol. 5, 261-268. Warranty These products are warranted to perform as described in their labeling and in CHEMICON literature when used in accordance with their instructions. THERE ARE NO WARRANTIES, WHICH EXTEND BEYOND THIS EXPRESSED WARRANTY AND CHEMICON DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE. CHEMICON s sole obligation and purchaser s exclusive remedy for breach of this warranty shall be, at the option of CHEMICON, to repair or replace the products. In no event shall CHEMICON be liable for any proximate, incidental or consequential damages in connection with the products. 2003-2005: CHEMICON International, Inc. - By CHEMICON International, Inc. All rights reserved. No part of these works may be reproduced in any form without permissions in writing. 10

Cat No. SGT440 January 2005 Revision C: 41444