181402 ASK1, GST-Fusion, Human, Recombinant, S. frugiperda

181402
  
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      Overview

      Replacement Information
      Description
      Overview

      This product has been discontinued.





      Recombinant, human ASK1 (accession no. NM_005923), consisting of amino acids 649-946, fused at the N-terminus to GST and expressed in S. frugiperda insect cells using a baculovirus expression system. ASK1, also known as MAPKKK5, activates MKK3, MKK4 (SEK1), and MKK6, is a serine/threonine kinase that is activated in response to TNFα. It plays a central role in stress-induced apoptosis; overexpression of ASK1 induces apoptotic cell death. The active kinase is useful in the study of ASK1 activity regulation and inhibitor screening.
      Catalogue Number181402
      Brand Family Calbiochem®
      SynonymsMEKK5, MAPKKK5
      Application Data

      The specific activity of ASK1 was measured as outlined above.
      References
      ReferencesNishitoh, H. et al.1998. Molec. Cell 2, 389.
      Ichijo, H. et al. 1997. Science 275, 90.
      Product Information
      Unit of DefinitionKinase activity is measured as the molar amount of phosphate incorporated into MBP at 30°C using a final concentration of 50 µM ATP.
      FormLiquid
      FormulationIn 150 mM NaCl, 50 mM Tris-HCl, 0.25 mM DTT, 0.1 mM EGTA, 0.1 mM EDTA, 0.1 mM PMSF, 25% glycerol, pH 7.5.
      Applications
      Biological Information
      Purity≥90% by SDS-PAGE
      Specific Activity≥95 nmol/min/mg
      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

      Certificates of Analysis

      TitleLot Number
      181402

      References

      Reference overview
      Nishitoh, H. et al.1998. Molec. Cell 2, 389.
      Ichijo, H. et al. 1997. Science 275, 90.
      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.

      Revision21-October-2010 JSW
      SynonymsMEKK5, MAPKKK5
      Application Data

      The specific activity of ASK1 was measured as outlined above.
      DescriptionRecombinant, human ASK1 (accession no. NM_005923), consisting of amino acids 649-946, fused at the N-terminus to GST and expressed in S. frugiperda insect cells using a baculovirus expression system. ASK1, also known as MAPKKK5, activates MKK3, MKK4 (SEK1), and MKK6, is a serine/threonine kinase that is activated in response to TNFα. It plays a central role in stress-induced apoptosis; overexpression of ASK1 induces apoptotic cell death. The active kinase is useful in the study of ASK1 activity regulation and inhibitor screening.
      FormLiquid
      FormulationIn 150 mM NaCl, 50 mM Tris-HCl, 0.25 mM DTT, 0.1 mM EGTA, 0.1 mM EDTA, 0.1 mM PMSF, 25% glycerol, pH 7.5.
      Concentration Label Please refer to vial label for lot-specific concentration
      Recommended reaction conditions
      Activity Assay Materials Required • Kinase Assay Buffer: 25 mM MOPS, 25 mM MgCl2, 12.5 mM β-glycerophosphate, 5 mM EGTA, 2 mM EDTA, pH 7.2; add 0.25 mM DTT just prior to use • Kinase Dilution Buffer: Kinase Assay Buffer diluted 1:5 with 50 ng/µl BSA • ASK1: Prepare serial dilutions using Kinase Dilution Buffer as outlined in the sample data below or as deemed necessary for individual experimental conditions • 10 mM ATP Stock Solution: Dissolve 55 mg ATP in 10 ml Kinase Assay Buffer; dispense into 200 µl aliquots and freeze (-20°C) • 250 µM [32P]-ATP Assay Cocktail: 150 µl 10 mM ATP stock solution, 100 µl [32P]-ATP (1 mCi/100 µl), 5.75 ml Kinase Assay Buffer; dispense into 1 ml aliquots and freeze (-20°C) • Substrate: Dissolve myelin basic protein (MBP) in dH2O to a final concentration of 1 mg/ml
      Activity Assay Protocol 1. Thaw an aliquot of [32P]-ATP Assay Cocktail in a shielded container in a designated radioactive working area. 2. Thaw the ASK1, Kinase Assay Buffer, Substrate, and Enzyme Dilution Buffer on ice. 3. In a pre-cooled tube, add the following components: • 10 µl diluted ASK1 • 10 µl 1 mg/ml Substrate stock 4. Prepare a Blank as outlined in step 3, replacing the 10 µl Substrate with an equal volume of water. 5. Initiate the reaction by adding 5 µl [32P]-ATP Assay Cocktail (final volume is 25 µl) and incubating in a water bath at 30°C for 15 min. 6. Terminate the reaction by spotting 20 µl of the reaction mixture onto individual pre-cut strips of phosphocellulose P81 paper. 7. Air-dry the pre-cut strips and sequenentially wash in 1% phosphoric acid with constant, gentle stirring for 10 min. Repeat for a total of 3 washes. 8. Count the radioactivity in presence of scintillation fluid. 9. Determine the corrected cpm by substracting the value of the Blank from each sample and calculate as follows:

      Figure 1: Calculation of Activity

      Purity≥90% by SDS-PAGE
      Specific activity≥95 nmol/min/mg
      Unit definitionKinase activity is measured as the molar amount of phosphate incorporated into MBP at 30°C using a final concentration of 50 µM ATP.
      Storage ≤ -70°C
      Avoid freeze/thaw
      Do Not Freeze Ok to freeze
      Special InstructionsFollowing initial thaw, aliquot and freeze (-70°C).
      Toxicity Standard Handling
      ReferencesNishitoh, H. et al.1998. Molec. Cell 2, 389.
      Ichijo, H. et al. 1997. Science 275, 90.