475701 | MKK6, GST-Fusion, Human, Recombinant, E. coli

475701
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      Overview

      Replacement Information

      Pricing & Availability

      Catalog NumberAvailability Packaging Qty/Pack Price Quantity
      475701-20UG
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          Plastic ampoule 20 μg
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          Description
          OverviewRecombinant, human MKK6 fused to GST at the N-terminus to GST and expressed in E. coli. MKK6 is a member of the dual specificity protein kinase family, which functions as a mitogen-activated protein (MAP) kinase kinase. It is suitable for labeling MAP kinase substrates.
          Catalogue Number475701
          Brand Family Calbiochem®
          SynonymsMEK6, Mitogen Activated Protein Kinase kinase 6, SAPKK3
          Application Data
          Increasing amounts of MKK6 were subjected to SDS-PAGE and the purity analyzed by Coomassie Blue staining.

          Increasing amounts of MKK6 were used to activate 10 µg p38α/SAPK2a and the activity of p38α/SAPK2a was measured as outlined in the protocols above.
          References
          ReferencesChabaud-Riou, M. and Firestein, G.S. et al. 2004. Am. J. Pathol. 164, 177.
          Zhu, X., et al. 2001. J. Neurochem. 79, 311.
          Product Information
          Unit of DefinitionOne unit is defined as the amount of enzyme that will activate p38alpha/SAPK2α (0.5 mg/ml) by 1 unit/min using 125 µM ATP at 30°C. One unit of p38α/SAPK2α activity is defined as the amount of enzyme that will transfer 1 pmol phosphate to myelin basic protein per min at 30°C.
          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≥ 90% by SDS PAGE
          Specific Activity≥ 50,000 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
          475701

          References

          Reference overview
          Chabaud-Riou, M. and Firestein, G.S. et al. 2004. Am. J. Pathol. 164, 177.
          Zhu, X., et al. 2001. J. Neurochem. 79, 311.
          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.

          Revision08-August-2008 RFH
          SynonymsMEK6, Mitogen Activated Protein Kinase kinase 6, SAPKK3
          Application Data
          Increasing amounts of MKK6 were subjected to SDS-PAGE and the purity analyzed by Coomassie Blue staining.

          Increasing amounts of MKK6 were used to activate 10 µg p38α/SAPK2a and the activity of p38α/SAPK2a was measured as outlined in the protocols above.
          DescriptionRecombinant, human MKK6 fused to GST at the N-terminus and expressed in E. coli. MKK6 is a member of the dual specificity protein kinase family, which functions as a mitogen-activated protein (MAP) kinase kinase. It is suitable for labeling MAP kinase substrates.
          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 p38α/SAPK2a 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 • p38&alpha/SPK2a (substrate): in 300 mM NaCl, 50 mM Tris-HCl, 2 mM Dtt, pH 8.5 •MKK6: 100-fold lower concentration than p38α/SAPK2, 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 p38α/SAPK2a and MKK6. 2. Add 1/8 volume Assay Buffer to a clean microcentrifuge tube on ice. 3. Add 3/8 volume H2O. 4. Add 1/8 volume MKK6. 5. Add 1/8 volume p38α/SAPK2a. 6. Add 1/4 volume Magnesium/ATP Mix. 7. Incubate at 30°C for 1 h. 8. Stop the reaction by placing the tubes on ice. 9. Assay the p38α/SAPK2a activity using the protocol outlined below. Note: The reaction may be dialyzed against 300 mM NaCl, 50 mM Tris-HCl, 2 mM DTT, pH 8.5, diluted with an equal volume of 99% glycerol, and stored at -70°C until use. 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 •p38α/SAPK2a, activated: 2.5-10 ng/µl, activated by MKK6 (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 2.5 µl Assay Buffer to a clean microcentrifuge tube on ice. 2. Add 10 µl Substrate. 3. Add 2.5 µl p38α/SAPK2a (25-100 ng/assay). 4. Add 5 µl Diluted [γ32P]ATP/Magnesium/ATP Mix. 5. Incubate at 30°C for 20 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≥ 90% by SDS PAGE
          Specific activity≥ 50,000 units/mg protein
          Unit definitionOne unit is defined as the amount of enzyme that will activate p38alpha/SAPK2α (0.5 mg/ml) by 1 unit/min using 125 µM ATP at 30°C. One unit of p38α/SAPK2α activity is defined as the amount of enzyme that will transfer 1 pmol phosphate to myelin basic protein 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
          ReferencesChabaud-Riou, M. and Firestein, G.S. et al. 2004. Am. J. Pathol. 164, 177.
          Zhu, X., et al. 2001. J. Neurochem. 79, 311.