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

475702
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      Overview

      Replacement Information

      Pricing & Availability

      Catalog NumberAvailability Packaging Qty/Pack Price Quantity
      475702-10UG
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          Plastic ampoule 10 μg
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          Description
          OverviewRecombinant, 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
          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

          SDS

          Title

          Safety Data Sheet (SDS) 

          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.