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475702 MEK1, GST-Fusion, Human, Recombinant, E. coli

475702
  
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      Overview

      Replacement Information
      Description
      Overview

      This product has been discontinued.



      Recombinant, human MEK1 fused to GST at the N-terminus and expressed in E. coli. MEK1 is a member of a dual-specificity protein kinase family that phosphorylates threonine and tyrosine residues on ERK 1 and 2.

      Catalogue Number475702
      Brand Family Calbiochem®
      SynonymsMAP Kinase Kinase, ERK Kinase 1, Microtubule-associated protein 2 kinase
      Application Data
      Increasing amounts of MEK1 were subjected to SDS-PAGE and the purity analyzed by Coomassie Blue staining.

      Increasing amounts of MEK1 were used to activate 10 µg ERK1 and the activity of ERK1 was analyzed as outlined in the activation and activity protocols above.
      References
      ReferencesAllen, L.F., et al. 2003. Semin. Oncol. 30, 105.
      Schramek, H., et al. 1997. J. Biol. Chem. 272, 11426.
      Alessandrini, A., et al. 1996. J. Biol. Chem. 271, 31612.
      Product Information
      Unit of DefinitionOne unit is defined as the amount of enzyme that activates ERK1 (0.3 mg/ml) by 1 unit/min using 100 µM ATP at 30°C. One unit ERK1 activity is defined as that amount of enzyme required to transfer 1 pmol phosphate to myelin basic protein, in the presence of 125 µM ATP, per min at 30°C.
      FormLiquid
      FormulationIn 270 mM sucrose, 150 mM NaCl, 50 mM Tris-HCl, 1 mM DTT, 0.1 mM EGTA, pH 7.5
      Quality LevelMQ100
      Applications
      Biological Information
      Purity≥80% by SDS PAGE
      Specific Activity≥ 250,000 units/mg protein
      Concentration Label Please refer to vial label for lot-specific concentration
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Storage and Shipping Information
      Ship Code Dry Ice Only
      Toxicity Standard Handling
      Storage ≤ -70°C
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Special InstructionsFollowing initial thaw, aliquot and freeze (-70°C).
      Packaging Information
      Transport Information
      Supplemental Information
      Specifications

      Documentation

      MEK1, GST-Fusion, Human, Recombinant, E. coli MSDS

      Title

      Safety Data Sheet (SDS) 

      MEK1, GST-Fusion, Human, Recombinant, E. coli Certificates of Analysis

      TitleLot Number
      475702

      References

      Reference overview
      Allen, L.F., et al. 2003. Semin. Oncol. 30, 105.
      Schramek, H., et al. 1997. J. Biol. Chem. 272, 11426.
      Alessandrini, A., et al. 1996. J. Biol. Chem. 271, 31612.
      Data Sheet

      Note that this data sheet 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.

      Revision02-January-2008 JSW
      SynonymsMAP Kinase Kinase, ERK Kinase 1, Microtubule-associated protein 2 kinase
      Application Data
      Increasing amounts of MEK1 were subjected to SDS-PAGE and the purity analyzed by Coomassie Blue staining.

      Increasing amounts of MEK1 were used to activate 10 µg ERK1 and the activity of ERK1 was analyzed as outlined in the activation and activity protocols above.
      DescriptionRecombinant, human MEK1 fused to GST at the N-terminus and expressed in E. coli. MEK1 is a member of a dual-specificity protein kinase family that phosphorylates threonine and tyrosine residues on ERK 1 and 2.
      FormLiquid
      FormulationIn 270 mM sucrose, 150 mM NaCl, 50 mM Tris-HCl, 1 mM DTT, 0.1 mM EGTA, pH 7.5
      Concentration Label Please refer to vial label for lot-specific concentration
      Recommended reaction conditions
      Kinase Activity Assay Activation of ERK1 Required Materials •Assay Buffer: 25 mM β-glycerophosphate, 20 mM Tris-HCl, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM DTT, pH 7.5 •ERK1 Dilution Buffer: 150 mM NaCl, 50 mM Tris-HCl, 2 mM DTT, pH 8.0 •ERK1 (substrate): in 150 mM NaCl, 50 mM Tris-HCl, 5 mM EDTA, 2 mM DTT, pH 8.0 •MEK1: 10-fold lower concentration that ERK1, diluted in Assay Buffer •Magnesium/ATP Mix: 75 mM MgCl2, 500 µM ATP Activation Protocol 1. Determine the reaction volume based on the concentration of ERK1 to be activated. 2. Add 1/5 volume Assay Buffer to a clean microcentrifuge tube on ice. 3. Add ERK1 to a final concentration of 300 µg/ml. 4. Add MEK1 at a concentration that is 1/10 that of ERK1. 5. Add ERK1 Dilution Buffer up to 4/5 volume. 6. Add 1/5 volume Magnesium/ATP Mix. 7. Incubate at 30°C for 30 min. 8. Stop the reaction by placing the tubes on ice. 9. Assay the ERK1 activity using the protocol outlined below. Note: The reaction may be dialyzed against 300 mM NaCl, 100 mM Tris-HCl, 2 mM DTT, pH 8.5, diluted with an equal volume of 99% glycerol, and stored at -70°C until use. This activation protocol can also be used for ERK2. Activity Assay Required Materials •Assay Buffer: 25 mM β-glycerophosphate, 20 mM Tris-HCl, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM DTT, pH 7.5 •Substrate: myelin basic protein, 2 mg/ml •ERK1, activated: 2.5-10 ng/µl, activated by MEK1 (above), diluted in Assay Buffer just prior to use •Magnesium/ATP Mix: 75 mM MgCl2, 500 µM ATP •Diluted [γ32P]ATP/Magnesium/ATP Mix: 197 µl Magnesium/ATP Mix, 3 µl (30 Ci) [γ32P]ATP (3000 Ci/mmol) Activity Protocol 1. Add 10 µl Assay Buffer to a clean microcentrifuge tube on ice. 2. Add 10 µl Substrate. 3. Add 2.5 µl ERK1 (25-100 ng/assay). 4. Add 10 µl Diluted [γ32P]ATP/Magnesium/ATP Mix. 5. Incubate at 30°C for 10 min. 6. Stop the reaction by placing the tubes on ice. 7. Spot 10 µl of each reaction on P81 paper and allow to bind for 30 s. 8. Wash by immersing the paper in 0.75% phosphoric acid and gently shaking. Repeat for a total of 3 washes. 9. Wash as in step 8 using acetone. 10. Dry using an infrared light. 11. Determine the cpm using a scintillation counter or imager.
      Purity≥80% by SDS PAGE
      Specific activity≥ 250,000 units/mg protein
      Unit definitionOne unit is defined as the amount of enzyme that activates ERK1 (0.3 mg/ml) by 1 unit/min using 100 µM ATP at 30°C. One unit ERK1 activity is defined as that amount of enzyme required to transfer 1 pmol phosphate to myelin basic protein, in the presence of 125 µM ATP, per min at 30°C.
      Storage ≤ -70°C
      Avoid freeze/thaw
      Do Not Freeze Ok to freeze
      Special InstructionsFollowing initial thaw, aliquot and freeze (-70°C).
      Toxicity Standard Handling
      ReferencesAllen, L.F., et al. 2003. Semin. Oncol. 30, 105.
      Schramek, H., et al. 1997. J. Biol. Chem. 272, 11426.
      Alessandrini, A., et al. 1996. J. Biol. Chem. 271, 31612.