MEK1 Assay Kit 1 Catalog # Lot # 16875

Transcription

1 MEK1 Assay Kit 1 Kit Components Assay Dilution Buffer (ADB), Catalog # Three vials, each containing 1.0ml of assay dilution buffer (20mM MOPS, ph 7.2, 25mM ß-glycerol phosphate, 5mM EGTA, 1mM sodium orthovanadate, 1mM dithiothreitol). Human Activated MEK1, Catalog # , Lot # Two vials, each containing 5 Units in 50ml of 50mM Tris-HCl, ph 7.4, 0.1mM EDTA, 50mM NaCl. 0.1% 2-mercaptoethanol and 5% glycerol. Mouse p42 MAP Kinase - GST (unactive), Catalog # , Lot # Four vials, each containing 12.5µg in 50µl of 50mM Tris-HCl, ph 7.5, 0.1mM EGTA, 0.03% Brij-35, 0.1% 2-mercaptoethanol and 50% glycerol. Magnesium/ATP Cocktail, Catalog # One vial containing 1.0ml of Mg 2+ /ATP cocktail (75mM magnesium chloride and 500µM ATP in ADB). P81 Phosphocellulose Squares, Catalog # One pouch containing 200 prelabeled squares. Kit Description Quantity: 50 assays per kit and sufficient MEK1 positive control kinase for 20 assays. Storage and Stability: Stable for 6 months at -70 C from date of shipment. Use: The assay kit is designed to measure the phosphotransferase activity in immunoprecipitates and column fractions. Crude cell lysates may also be used but detergents/biochemicals contained in the cell lysis buffer may inhibit MEK1 and p42 MAP kinase activity. The kinase cascade reaction uses a recombinant unactive p42 MAPK-GST, which is phosphorylated and activated by Human activated MEK1. Activated p42 MAPK-GST then phosphorylates a specific substrate, myelin basic protein (MBP). The phosphorylated MBP is separated from the residual [γ- 32 P]ATP by differential binding to P81 phosphocellulose paper, which must be extensively washed with phosphoric acid before determination of bound radioactivity by liquid scintillation counting. The assay is linear for incubation times of up to 30 minutes and incorporation of up to 20% of total ATP. Longer incubation or higher levels of incorporation may not be linear and therefore may not accurately measure MEK1 kinase activity in the sample extract. The enzyme assay is rapid, convenient and fairly specific for MEK1. Each kit contains sufficient reagents for 50 individual MEK1 kinase assays. There is enough MEK1 provided for 20 positive control assays. Activated MEK1, Mg 2+, 30 C, 30 min Unactive MAP Kinase + ATP Active MAP Kinase + ADP Active MAP Kinase, Mg 2+,30 C, 10 min MBP + [γ- 32 P]ATP [ 32 P]-MBP + ADP FOR IN VITRO RESEARCH USE ONLY NOT RECOMMENDED OR INTENDED FOR DIAGNOSIS OF DISEASE IN HUMANS OR ANIMALS DO NOT USE IN HUMANS OR IN ANIMALS

2 Page Two of Five Technical Information for Kit Components Human Activated MEK1 (recombinant enzyme produced in E. coli ) Product Description: Recombinant human activated MEK1 fused with GST at the N terminus (71kDa fusion protein) and His 6 at the C terminus which is expressed in E coli. The enzyme has been purified >75% by chromatography over Glutathione-Sepharose chromatography and nickel agarose. Activity: 1Unit of MEK1 will activate 1Unit of unactive p42 MAP Kinase-GST which in turn incorporates 1nmol/min of phosphate into myelin basic protein (MBP). Background: MAP Kinase Kinase or Erk Kinase (MEK) are a unique class of kinases, characterized by an extremely narrow substrate specificity (MAP Kinase or Erk), and dual specificity for threonine and tyrosine, represented by a Thr-Glu- Tyr motif in MAP Kinase. MEK can be phosphorylated in vitro by several kinases, including protein kinase C, p34 cdc2, and MAP Kinase itself, but is activated only on phosphorylation of serines 218 and 222, which can be carried out by the Raf family members, Mos, or less efficiently, by the mammalian homolog of STE7 MEK Kinase. 1-3 Together with Raf, MEK-1 can associate with activated Ras, 4,5 and can be inactivated by p34 cdc2 phosphorylation. 6 Activity of MEK can be assessed by phosphorylation of unactive MAP Kinase, or by activation of MAP Kinase in an assay coupled to phosphorylation of MBP, which serves as a MAP Kinase substrate. Finally, because so many kinases are able to phosphorylate MEK, assays of MEK activators should be carried out by measuring MEK activation rather than MEK phosphorylation alone. Mouse p42 MAP Kinase - GST (unactive) (recombinant enzyme produced in E. coli ) Product Description: Recombinant mouse p42 MAP Kinase fused with GST at the N terminus (62 kda fusion protein) and expressed in E coli. The enzyme has been purified >90% by Glutathione-Sepharose chromatography. Specific Activity: The enzyme preparation demonstrates no endogenous kinase activity. When maximally activated with MEK (MAPK Kinase), the p42 MAP Kinase exhibits a specific activity of 1300U/mg. 1U = 1nmol phosphate incorporated into myelin basic protein (MBP) per minute. Specificity: Recognizes the phosphorylation site motif: Pro-X-(Ser/Thr)-Pro, where X is ideally a basic or neutral amino acid residue. MAPK can phosphorylate a range of proteins in vitro including p90 rsk, p70 S6 kinase, p74 raf1, EGF receptor, Myc, and Jun. Background: An important convergence point involved in the signal transduction pathways of many different growth factors, hormones, and cytokines is a family of 41-44kDa serine/threonine kinases collectively called either MAPKs (for mitogen-activated protein kinases) or Erks (for extracellular-regulated kinases). MAPK is activated by sequential phosphorylation on both tyrosine and threonine residues by the dual tyrosine/threonine kinase MEK (MAPK Kinase). The serine/threonine kinase Raf in turn can phosphorylate and activate MEK during intracellular signaling. Activation of MAPK is directly regulated by a specific MAPK phosphatase and indirectly regulated by protein kinase A activation which results in inhibition of Raf activity in mammalian cells. References: 1. Alessi, D.R., et al., Methods Enzymol 255: , Alessi, D.R., et al., EMBO J. 13: , Marshall, C.J., Curr. Op. Genet. Dev. 4: 82-89, Van Aelst, L., et al., Proc. Natl. Acad. Sci. USA 90: , Jelinek, T., et al., Mol. Cell. Biol. 14: , Rossomando, A.J., et al., Mol. Cell. Biol. 14: , 1994.

3 Page Three of Five Other components required but not included as part of kit are: Myelin Basic Protein (Catalog #13-104) Cell or tissue extract containing active MEK1 acetone vortex mixer plexiglass shielding shaking incubator timer variable volume (5-200µl) pipet + tips phosphoric acid scintillation vials scintillation fluid scintillation counter [γ- 32 P]ATP Ci/mmol, obtained from DuPont-New England Nuclear, Catalog NEG- 002A. Safety Warnings and Precautions: The MEK1 kinase assay kit is designed for research use only and not recommended for internal use in humans or animals. Since the kit involves the use of radioactive [γ- 32 P]ATP, please follow your institutional instructions for handling, use, storage and disposal of radioactive materials. All chemicals should be considered potentially hazardous and the principles of good laboratory practice should be followed. MEK1 Assay Kit Procedures All components should be thawed, thoroughly mixed before beginning the assay and stored on ice. In particular, assay dilution buffer (ADB) and Magnesium/ATP cocktail must be rapidly thawed and mixed completely before proceeding with assay. Do not use extended thawing time. The assay components can be refrozen at -20 C for extended periods. Perform all pre-incubation reactions at 1 C over an ice bath. The kinase assay can also be performed at room temperature but will not give linear results. In the first stage, aliquot 20µl of ADB, 10µl of cold Magnesium/ATP cocktail, 0.5Units of activated MEK1 or an experimental sample containing active MEK1 and 1µg of unactive MAP kinase in the bottom of a microcentrifuge tube. Incubate the reaction by shaking at 30 C for 30 minutes. Remove 4µl of this mixture and add to the second stage component mixture. To prepare the second stage component mixture, add 10µl of ADB, 10µl of MBP substrate and 4µl from the first stage to the microcentrifuge tube. Add 10µl of the diluted [γ- 32 P]ATP mixture prepared as described below under stock solutions, pulse spin the microcentrifuge tube, and incubate in a shaking incubator for 10 minutes at 30 C. Stop the reaction by removing 25µl of the reaction mixture and transferring the sample slowly onto the center of a P81 phosphocellulose paper. Allow the radiolabeled substrate to bind to the filter paper for 30 seconds before immersing the paper into a beaker containing 0.75% phosphoric acid. Wash the papers thoroughly in the beaker with up to 10 rinses of 0.75% phosphoric acid. Do not wash more than 30 papers per beaker. Dispose of each rinse according to local radioisotope regulations. After washing, add acetone and wash for 2 minutes, remove the paper with forceps and transfer the paper into a 5ml scintillation vial. Add scintillation cocktail and quantitate the bound radioactivity on the paper in a scintillation counter. Alternatively, assay squares may be washed in a 50ml conical tube containing 40ml 0.75% phosphoric acid. Gently shake the assay squares for 5 minutes on a rotator. Discard the wash in a liquid radioisotope waste container and repeat the wash step twice. Wash the squares in 20ml of acetone for 3 minutes and process the squares for liquid scintillation counting. Suitable blanks should always be performed to correct for non-specific binding of [γ- 32 P]ATP and its breakdown products to the phosphocellulose paper. Controls for endogenous phosphorylation of proteins in the sample extract can be performed by substituting assay dilution buffer for substrate cocktail.

4 Page Four of Five Kinase Assay Protocol Stock Solutions: 1. Assay Dilution Buffer (ADB): 20mM MOPS, ph 7.2, 25mM ß-glycerol phosphate, 5mM EGTA, 1mM sodium orthovanadate, 1mM dithiothreitol. 2. Magnesium/ATP Cocktail: 75mM magnesium chloride and 500µM ATP in ADB. 3. [γ- 32 P]ATP: Stock 1mCi/100µl (3000Ci/mmol, obtained from DuPont-NEN). Make 10µl aliquots (100mCi/vial). Before starting the assay, dilute an aliquot with 90µl Magnesium/ATP cocktail. 4. Human Activated MEK1: Use U per positive control. 5. MBP Substrate: Prepare a 2mg/ml stock (Catalog #13-104) by diluting with ADB. 6. GST-p42 unactive MAP Kinase: Use at 1.0µg per assay point. Assay Procedure: First Stage: Activation of GST-p42 unactive MAP Kinase 1. Add 20µl of ice cold ADB/assay. 2. Add 10µl cold Magnesium/ATP cocktail. 3. Add 1-5µl of activated MEK1 ( U in assay dilution buffer); immunoprecipitated MEK1 as well as cell or tissue extracts may be used. 4. Add 4µl of unactive GST-p42 MAP Kinase (1µg/assay). 5. Incubate for 30 minutes at 30 C in a shaking incubator. 6. Remove 4ml of this mixture and add to Second Stage component mixture. Second Stage: Assay of GST-p42 MAP Kinase Activity Via Phosphorylation of MBP 1. Add 10µl of ADB. 2. Add 10µl of MBP substrate. (20µg) 3. Add 10µl cold adenosine 5-triphosphate and [γ- 32 P]ATP mixture. 4. Use a microcentrifuge pulse to collect the components in the bottom of the microcentrifuge tube. 5. Incubate for 10 minutes at 30 C. 6. Spot 25µl on a 2cm x 2cm P81 paper. 7. Wash assay squares three times with 0.75% phosphoric acid for five minutes per wash. 8. Wash assay squares once with acetone for five minutes. 9. Transfer assay squares to a scintillation vial and add 5ml scintillation cocktail. Read in scintillation counter. Compare CPM of enzyme samples to CPM of control samples that contain no enzyme (background control). 10. As an alternative to scintillation counting, the first stage phosphorylation reaction may be performed with the diluted [γ- 32 P]ATP. A sample of the radiolabeled reaction mixture may then be analyzed by a combination of SDS-PAGE and autoradiography to determine MEK1 activity qualitatively by the visualization of MEK1 and MAPK.

5 Page Five of Five Activation of p42map Kinase-GST by MEK1 Data: The MEK1 assay kit was used to activate unactive p42map Kinase- GST. The kinase activity of p42map Kinase-GST was determined by using the MAP kinase assay kit (Catalog #17-133). This kit uses myelin basic protein (MBP) as a kinase substrate and contains a separate inhibitor cocktail, which blocks the activity of other serine/threonine kinases such as protein kinase A, protein kinase C, and calmodulin dependent kinases. The test results using a coupled phosphorylation assay are shown below: Activating Kinase Kinase Substrate 1 Kinase Substrate 2 Mean CPM Comments Activated MEK1, 0.5U none MBP 5,768 Background none Activated MEK1, 0.5U p42 MAPK-GST, unactive p42 MAPK-GST. unactive MBP 5,833 Background MBP 567,845 MEK1-dependent MAPK Activity