325897 Cdk1/CycB, GST-Fusion, Human, Recombinant, S. frugiperda

325897
  
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      Přehled

      Replacement Information
      Description
      OverviewRecombinant, human Cdk1 (amino acids 1-297) and recombinant human cyclin B (amino acids 1-433), each fused to a GST-His6thrombin cleavage site sequence at their respective N-termini and co-expressed in S. frugiperda insect cells using a baculovirus expression system. MW: 63882 (Cdk1) and 78925 (cyclinB).
      Catalogue Number325897
      Brand Family Calbiochem®
      References
      ReferencesJi, J.Y., et al. 2005. Development 132, 2501.
      Product Information
      Unit of DefinitionOne unit is defined as the amount of enzyme required to transfer 1 nmol phosphate to Rb-CTF per min at 30°C, using variable concentrations of ATP (100-759 nM).
      EC number2.7.1.112
      FormLiquid
      FormulationIn 100 mM NaCl, 50 mM Tris-HCl, 5 mM DTT, 4 mM reduced glutathione, 20% glycerol, pH 8.0.
      Applications
      Biological Information
      Specific Activity≥2 U/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

      Cdk1/CycB, GST-Fusion, Human, Recombinant, S. frugiperda Certificates of Analysis

      TitleLot Number
      325897

      References

      Přehled odkazů
      Ji, J.Y., et al. 2005. Development 132, 2501.
      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.

      Revision10-September-2007 JSW
      DescriptionRecombinant, human Cdk1 (amino acids 1-297) and recombinant human cyclin B (amino acids 1-433), each fused to a GST-His6thrombin cleavage site sequence in their respective N-termini and co-expressed in S. frugiperda insect cells using a baculovirus expression system. MW: 63882 (Cdk1) and 78925 (cyclinB).
      FormLiquid
      FormulationIn 100 mM NaCl, 50 mM Tris-HCl, 5 mM DTT, 4 mM reduced glutathione, 20% glycerol, pH 8.0.
      Concentration Label Please refer to vial label for lot-specific concentration
      Recommended reaction conditions
      96-Well Membrane Binding Kinase Activity Assay Protocol:
      Materials Required Standard Kinase Assay Buffer: 125 mM HEPES-NaOH, 7.5 mM MgCl2, 7.5 mM MnCl2, 7.5 µM sodium -orthovanadate, 2.5 mM DTT, pH 7.5 10X Kinase Dilution Buffer: 500 mM HEPES-NaOH, 2.5 mg/ml PEG20.000, 10 mM DTT, pH 7.5 Substrate: Rb-CTF, 2.5 µg/50 µl final concentration CDK1/CycB, GST-Fusion, Human, Recombinant, S. frugiperda, diluted in 1X Kinase Dilution Buffer to desired concentration (we use 100 ng in a 50 µl reaction) 33P-γ-ATP ATP Mix: 1 µM ATP in H2O with 1 x 106 cpm/5 µl 96-well V-bottom MTP (polypropylene) plates (Nunc Cat. No. 442587) 96-well Multiscreen Vacuum Manifold (Millipore Cat. No. MAVM096OR) 96-well Multiscreen Filterplates, phosphocellulose membrane (Millipore Cat. No. MSPH N6B50) and glass fiber (Millipore Cat. No. MSFC N6B50) 2% H3PO4 0.9% NaCl
      Activity Assay Protocol 1. In a polypropylene V-bottom plate add 20 µl Kinase Assay Buffer 2. If desired, add 5 µl test substance (e.g., inhibitor, activator, etc.; in 10% DMSO) 3. Add 10 µl Substrate (diluted in H2O). 4. Add 10 µl diluted CDK1/CycB. 5. Add 5 µl ATP Mix. 6. Mix on a shaker and incubate at 30°C for 80 min. 7. Stop the reaction with 50 µl H3PO4. 8. Prewet the filter plate with 200 µl H2O. 9. Filter the reaction mix by applying vacuum. 10. Wash 3 times with 200 µl 0.9% NaCl. 11. Add 200 µl scintillation fluid to each well and count on a scintillation counter.
      EC number2.7.1.112
      Specific activity≥2 U/mg protein
      Unit definitionOne unit is defined as the amount of enzyme required to transfer 1 nmol phosphate to Rb-CTF per min at 30°C, using variable concentrations of ATP (100-759 nM).
      Storage ≤ -70°C
      Avoid freeze/thaw
      Do Not Freeze Ok to freeze
      Special InstructionsFollowing initial thaw, aliquot and freeze (-70°C).
      Toxicity Standard Handling
      ReferencesJi, J.Y., et al. 2005. Development 132, 2501.