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CBA099 K-LISA™ PDK2 Activity Kit

K-LISA™ PDK2 Activity Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
CBA099
  
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      Overview

      Replacement Information
      Description
      Overview

      This product has been discontinued.





      Designed for rapid in vitro screening of PDK2 inhibitors and for assessing activity of purified PDK2 enzyme candidates.
      Catalogue NumberCBA099
      Brand Family Calbiochem®
      Application Data

      *If no inhibitor is to be included, up to 20 µl sample can be added or inhibitor can be substituted with 10 µl dH2O.

      The activity of increasing concentrations of PDK2 enzymes, PKCβII (Cat. No. 539657) and MAPKAP2 (Cat. No. 475861), were assayed in the absence of inhibitors as outlined in the Detailed Protocol above.

      The activity of PKCβII (Cat. No. 539657) was measured in the presence of increasing concentrations of a specific PKCβII Inhibitor (left panel) and Staurosporine (right panel) as outlined in the Detailed Protocol above.
      Materials Required but Not Delivered• ELISA plate reader with 450 nm filter, preferably in the dual wavelength mode with a reference filter at 540-600 nm filter.
      • Wash bottle or multichannel dispenser for washing.
      • 30°C incubator
      References
      ReferencesBhaskar, P.T., et al. 2007. Dev. Cell 12, 487.
      Tsurutani, J., et al. 2006. J. Clin. Oncol. 24, 306.
      Sarbassov, D.D., et al. 2005. Science 307, 1098.
      Woodgeff, J.R., et al. 2005. Curr. Opin. Cell Biol. 17, 150.
      Hanada, M., et al. 2004. Biochim. Biophys. Acta 1697, 3.
      Kreisberg, J.I., et al. 2004. Biochim. Biophys. Acta 64, 5232.
      Product Information
      Detection methodColorimetric
      Form96 Tests
      Format96-well plate
      Kit containsNi-NTA Coated 96-well Plate, Anti-Akt (pSer⁴⁷³) Mouse mAb, inactive His-Akt1, 100X Inhibitor, Kinase Assay Buffer, 5X ATP/MgCl₂ Mix, Antibody Diluent, Kinase Stop Solution, TMB Substrate, HRP-Conjugate, ELISA 20X Plate Wash Concentrate, ELISA Stop Solution, Plate Sealers, and a user protocol.
      Applications
      Biological Information
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 20/21/22-25-35-36/37/38-61

      Harmful by inhalation, in contact with skin and if swallowed.
      Toxic if swallowed.
      Causes severe burns.
      Irritating to eyes, respiratory system and skin.
      May cause harm to the unborn child.
      S PhraseS: 24/25-26-36/37/39-45

      Avoid contact with skin and eyes.
      In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
      Wear suitable protective clothing, gloves and eye/face protection.
      In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
      Product Usage Statements
      Intended useThe Calbiochem® K-LISA™ PDK-2 Activity Kit is designed for rapid in vitro screening of PDK2 inhibitors and for assessing activity of purified PDK2 enzyme candidates.
      Storage and Shipping Information
      Ship Code Multiple Storage Conditions
      Toxicity Multiple Toxicity Values, refer to MSDS
      Hazardous Materials Attention: Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.
      Storage Multiple storage requirements
      Storage ConditionsUpon arrival store the PDK2 Standard (Recombinant PKCβII) at -70°C, the Ni-NTA Plate at 4-20°C, and all remaining components at -20°C.
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsNi-NTA Coated 96-well Plate, Anti-Akt (pSer⁴⁷³) Mouse mAb, inactive His-Akt1, 100X Inhibitor, Kinase Assay Buffer, 5X ATP/MgCl₂ Mix, Antibody Diluent, Kinase Stop Solution, TMB Substrate, HRP-Conjugate, ELISA 20X Plate Wash Concentrate, ELISA Stop Solution, Plate Sealers, and a user protocol.
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      CBA099 0

      Documentation

      K-LISA™ PDK2 Activity Kit Certificates of Analysis

      TitleLot Number
      CBA099

      References

      Reference overview
      Bhaskar, P.T., et al. 2007. Dev. Cell 12, 487.
      Tsurutani, J., et al. 2006. J. Clin. Oncol. 24, 306.
      Sarbassov, D.D., et al. 2005. Science 307, 1098.
      Woodgeff, J.R., et al. 2005. Curr. Opin. Cell Biol. 17, 150.
      Hanada, M., et al. 2004. Biochim. Biophys. Acta 1697, 3.
      Kreisberg, J.I., et al. 2004. Biochim. Biophys. Acta 64, 5232.
      User Protocol

      Revision16-July-2008 JSW
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      StorageUpon arrival store the PDK2 Standard (Recombinant PKCβII) at -70°C, the Ni-NTA Plate at 4-20°C, and all remaining components at -20°C.
      Intended useThe Calbiochem® K-LISA™ PDK-2 Activity Kit is designed for rapid in vitro screening of PDK2 inhibitors and for assessing activity of purified PDK2 enzyme candidates.
      BackgroundUnderstanding the regulation of Akt has been of major interest in elucidating the control of normal cellular physiology and malignant transformation. The serine/threonine kinase, Akt, also known as protein kinase B (PKB), plays key regulatory roles in diverse cellular processes including glucose metabolism, cell cycle progression, and apoptosis. Akt consists of an N-terminal pleckstrin homology (PH) domain, a central kinase domain, and a C-terminal regulatory hydrophobic motif (HF) domain. The PH domain interacts with membrane lipid products generated by PI 3-kinase, thereby releasing the autoinhibitory function of this domain and allowing phosphorylation of Thr308 in the central kinase domain by PDK1 (phospho-inositide-dependent kinase 1). PDK2 phosphorylates Ser473 in the HF domain, however the identity of PDK2 is not firmly established. Phosphorylation at Ser473 stabilizes the active configuration of Akt. So far at least eight kinases have been shown to function as a PDK2, i.e., catalyze the phosphorylation of Ser473: MAPKAP kinase 2, p38 MAP kinase, PKCα, PKCβII, DNA-PKCs, NEK6, integrin-linked kinase (ILK), and TORC2. However, some of these kinases might not play an important role as PDK2 in vivo.
      Principles of the assayThe K-LISA™ PDK2 Activity Kit is an ELISA-based activity assay that utilizes full-length, inactive His-Akt1 as substrate. The assay is useful for screening of inhibitors affecting the phosphorylation of the PDK2 site in Akt and for PDK2 activity assessment of various novel kinases. The His-Akt1 and PDK2 enzymes (e.g. PKCβII, supplied as a standard) are incubated in the presence or absence of PDK2 inhibitors in the wells of the Ni-NTA-Coated 96-Well Plate. The phosphorylated Akt substrate is detected using Anti-Akt (pSer⁴⁷³) monoclonal antibody followed by the HRP-Conjugate and color development with TMB Substrate. ELISA Stop Solution is used to stop the color development and the absorbance is read at 450 nm preferably with a reference wavelength of 540-600 nm. The absorbance is directly related to the level of PDK2 activity. Recombinant PKCβII is provided as a PDK2 Standard. Staurosporine serves as a positive control for inhibitor analysis. Inhibition profiles can be created based on PDK2 activity in the presence and absence of inhibitor compounds.
      Materials provided• Ni-NTA-Coated 96-Well Plate (Kit Component No. JA9444-1EA): 1 transparent plate, 96-wells, supplied as twelve 8-well strips coated with Ni-NTA
      • PDK2 Standard (recombinant PKCβII) (Kit Component No. JA9539-2UG): 1 vial, 2 µg, please refer to the vial label for lot-specific concentration
      • Anti-Akt (pSer473) mAb (Kit Component No. JA9537-11ML): 1 bottle, 11 ml, ready-to-use
      • His-Akt1, Inactive (Kit Component No. JA9538-12UG): 1 vial, 12 µg
      • 100X Inhibitor (Kit Component No. JA7915-25UL): 1 vial, 25 µl staurosporine, supplied as a 100X solution in DMSO
      • 5X Kinase Assay Diluent (Kit Component No. JA9540-5ML): 1 bottle, 5 ml, supplied as a 5X solution
      • 5X ATP/MgCl2 (Kit Component No. JA7914-2ML): 1 vial, 2 ml, supplied as a 5X solution
      • 400X HRP-Conjugate (Kit Component No. JA7643-100UL): 1 vial, 100 µl, supplied as a 400X solution
      • Kinase Stop Solution (Kit Component No. KP31517-5ML): 1 vial, 5 ml
      • Antibody Diluent (Kit Component No. JA7917-25ML): 1 bottle, 25 ml
      • ELISA 20X Plate Wash Concentrate (Kit Component No. JA1617-100ML): 1 bottle, 100 ml
      • TMB Substrate (Kit Component No. JA1608-12ML): 1 bottle, 12 ml, ready-to-use
      • ELISA Stop Solution (Kit Component No. JA1616-12ML): 1 bottle, 12 ml 2.5 N H₂SO₄, ready-to-use
      • Plate Sealers: 2 each
      Materials Required but not provided• ELISA plate reader with 450 nm filter, preferably in the dual wavelength mode with a reference filter at 540-600 nm filter.
      • Wash bottle or multichannel dispenser for washing.
      • 30°C incubator
      Reagent preparationPrepare all reagents immediately prior to use. The following table provides reagent preparation instructions to obtain the volume of Working Solutions (WS) required for one 8-well strip.

      Table 1: Preparation of Reagents
      Detailed protocol1. Remove required number of strips from the Ni-NTA-Coated 96-Well Plate and place them in the 96-well frame. Return the unused strips to the foil pouch, reseal the entire edge with tape, and store at 4°C.
      2. Add the following components to designated wells in the specified order:

      Table 2: Addition of Assay Components

      *If no inhibitor is to be included, up to 20 µl sample can be added or inhibitor can be substituted with 10 µl dH2O.


      3. Cover the plate with a Plate Sealer, mix the contents of the wells with a plate shaker or equivalent, and incubate for 60 min 30°C.
      4. Stop the kinase reaction by adding 10 µl Kinase Stop Solution to each well. (alternatively, the reaction may also be stopped by discarding the contents of the wells).
      5. Discard the contents of the wells by shaking the plate over the sink and wash the plate 3-4 times with 1X ELISA Plate Wash, 200 µl/well. Shake out the wash solution and dry the wells by tapping the inverted plate on paper towels.
      6. Add 100 µl Anti-Akt (pSer473) to each well and incubate for 1 h at room temperature.
      7. Wash the plate as described in step 5 above.
      8. Add 100 µl HRP-Conjugate WS to each well and incubate for 1 h at room temperature.
      9. Wash the plate as described in step 5 above.
      10. Add 100 µl TMB Substrate to each well and incubate for 10-15 min at room temperature.
      11. Add 100 µl ELISA Stop Solution to each well and read the absorbance to each well at 450 nm, preferably with a reference wavelength set at 540-600 nm.
      Assay characteristics and examples

      Figure 1: Enzyme Activity of Purified Recombinant PKCβII and MAPKAP2

      The activity of increasing concentrations of PDK2 enzymes, PKCβII (Cat. No. 539657) and MAPKAP2 (Cat. No. 475861), were assayed in the absence of inhibitors as outlined in the Detailed Protocol above.


      Figure 2: Inhibition by a Specific PKCβII inhibitor and Staurosporine

      The activity of PKCβII (Cat. No. 539657) was measured in the presence of increasing concentrations of a specific PKCβII Inhibitor (left panel) and Staurosporine (right panel) as outlined in the Detailed Protocol above.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      K-LISA™ and Interactive Pathways™ are trademarks of EMD Chemicals, Inc.