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CBA044 K-LISA™ IKKβ Inhibitor Screening Kit

K-LISA™ IKKβ Inhibitor Screening Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.

Synonyms: I-kappa-B-kinase beta screening kit, NFKBIKB screening kit, Nuclear factor NF-kappa-B inhibitor kinase beta screening kit, I-kappa-B kinase 2 screening kit, IKK2 screening kit, IκBKB screening kit, Inhibitor of nuclear factor kappa B kinase beta subunit screening kit

CBA044
  
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      Overview

      Replacement Information

      Key Spec Table

      Detection Methods
      Colorimetric
      Description
      OverviewThe K-LISA™ IKKβ-Inhibitor Screening Kit is designed for rapid in vitro screening of IKKβ inhibitors. The assay is an ELISA-based activity assay that utilizes a 50-amino acid GST-IκBα fusion polypeptide substrate that includes the Ser32 and Ser36 IKKβ phosphorylation sites. The GST-IκBα substrate and IKKβ are incubated in the presence of IKKβ inhibitors in the wells of a Glutathione-Coated 96-Well Plate, which allows for substrate phosphorylation and capture in a single step. The phosphorylated GST-IκBα substrate is detected using an Anti-Phospho IκBα (Ser32/Ser36) 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 IKKβ activity.
      Catalogue NumberCBA044
      Brand Family Calbiochem®
      SynonymsI-kappa-B-kinase beta screening kit, NFKBIKB screening kit, Nuclear factor NF-kappa-B inhibitor kinase beta screening kit, I-kappa-B kinase 2 screening kit, IKK2 screening kit, IκBKB screening kit, Inhibitor of nuclear factor kappa B kinase beta subunit screening kit
      Materials Required but Not Delivered• Spectrophotometer capable of measuring absorbance in 96-well plates at 450 nm, preferably in the dual wavelength mode with a reference wavelength of 540-600 nm.
      References
      ReferencesGreten, F.R., et al. 2004. Cell 118, 285.
      Baldwin, A.S., 2001. J. Clin. Invest. 107, 241.
      Yamamoto, Y. and Gaynor, R.B., 2001. J. Clin. Invest. 107, 135.
      Karin, M., et al. 2000. Annu. Rev. Immunol. 18, 621.
      Zandi, E., et al. 1997. Cell 91, 243.
      Product Information
      Detection methodColorimetric
      Form96 Tests
      Format96-well plate
      Kit containsGlutathione Coated 96-Well Plate, GST-IκBα (IKKβ Substrate), Anti-Phospho IκBα (Ser32/Ser36), IKKβ, Antibody Diluent, Kinase Assay Buffer, ATP/MgCl₂ Mix, IKKβ Inhibitor 500X, Kinase Stop Solution, ELISA 20X Plate Wash Concentrate, HRP-Conjugate, TMB Substrate, ELISA Stop Solution, Kinase Stop Solution, Plate Sealers, and a user protocol.
      Applications
      Biological Information
      Assay range5-10 ng
      Sample TypePurified enzyme
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe K-LISA™ IKKΒ-Inhibitor Screening Kit is designed for rapid in vitro screening of IKKΒ inhibitors.
      Storage and Shipping Information
      Ship Code Dry Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage Multiple storage requirements
      Storage ConditionsUpon arrival store the IKKΒ, His·Tag®, Human, Recombinant, Spodoptera frugiperda at -70°C and the remaining components at -20°C.
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsGlutathione Coated 96-Well Plate, GST-IκBα (IKKβ Substrate), Anti-Phospho IκBα (Ser32/Ser36), IKKβ, Antibody Diluent, Kinase Assay Buffer, ATP/MgCl₂ Mix, IKKβ Inhibitor 500X, Kinase Stop Solution, ELISA 20X Plate Wash Concentrate, HRP-Conjugate, TMB Substrate, ELISA Stop Solution, Kinase Stop Solution, Plate Sealers, and a user protocol.
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      CBA044 0

      Documentation

      K-LISA™ IKKβ Inhibitor Screening Kit Certificates of Analysis

      TitleLot Number
      CBA044

      References

      Reference overview
      Greten, F.R., et al. 2004. Cell 118, 285.
      Baldwin, A.S., 2001. J. Clin. Invest. 107, 241.
      Yamamoto, Y. and Gaynor, R.B., 2001. J. Clin. Invest. 107, 135.
      Karin, M., et al. 2000. Annu. Rev. Immunol. 18, 621.
      Zandi, E., et al. 1997. Cell 91, 243.
      User Protocol

      Revision24-January-2011 RFH
      SynonymsI-kappa-B-kinase beta screening kit, NFKBIKB screening kit, Nuclear factor NF-kappa-B inhibitor kinase beta screening kit, I-kappa-B kinase 2 screening kit, IKK2 screening kit, IκBKB screening kit, Inhibitor of nuclear factor kappa B kinase beta subunit screening kit
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      StorageUpon arrival store the IKKΒ, His·Tag®, Human, Recombinant, Spodoptera frugiperda at -70°C and the remaining components at -20°C.
      Intended useThe K-LISA™ IKKΒ-Inhibitor Screening Kit is designed for rapid in vitro screening of IKKΒ inhibitors.
      BackgroundIκB kinases, IKKα (also called IKK-1) and IKKβ (IKK-2), are related kinases that play a major role in the activation and regulation of the transcription factor, NF- κB. They are induced by stimuli such as TNFα and IL-1 to phosphorylate Ser32 and Ser36 in IκBα, the regulatory subunit of NF-κB. The phosphorylation of Iκα results in ubiquitination and subsequent degradation in the proteasome. The induced proteolysis of IκBα unmasks the nuclear localization signal within NF-κB, resulting in its rapid migration into the nucleus where it activates the transcription of numerous target genes. The IκB kinase (IKK) complex is composed of three polypeptides: the catalytic subunits, IKKα (IKK-1) and IKKβ (IKK-2), and the regulatory subunit, IKKγ (IKK-3/NEMO). IKKβ appears to be the principal kinase, whereas IKKα is not required for activation of IKK and degradation of IκBα by proinflammatory stimuli. IKKβ triggers the activation of NF-κB in response to infectious agents and proinflammatory cytokines, making it an attractive drug target for the treatment of inflammatory diseases. For example, it is reported that orally administered small molecule inhibitors of IKKβ lead to reduced incidence and severity of arthritis in a mouse model. In addition, NF-κB is over-expressed or constitutively activated in many cancer cells where it induces theexpression of anti-apoptotic genes, which correlates with resistance to anticancer therapies. Deletion of IKKβ in intestinal epithelial cells does not result in a decrease inflammation; it leads to a dramatic decrease in tumor incidence without affecting tumor size. This may be linked to increased epithelial apoptosis during tumor promotion. Deleting IKKβ in myeloid cells, however, results in a significant decrease in tumor size. This deletion diminishes expression of pro-inflammatory cytokines that may serve as tumor growth factors, without affecting apoptosis. Thus, specific inactivation of the IKK/NF-κB pathway in two different cell types can attenuate formation of inflammation-associated tumors in addition to suppressing apoptosis in advanced tumors. Therefore, inhibitors of IKKβ represent potential therapeutics for the treatment of both cancer and inflammation.
      Principles of the assayThe K-LISA™ IKKβ-Inhibitor Screening Kit is an ELISA-based activity assay that utilizes a 50-amino acid GST-IκBα fusion polypeptide substrate that includes the Ser³² and Ser³⁶ IKKβ phosphorylation sites. The GST-IκBα substrate and IKKβ His·Tag®, Human, Recombinant, Spodoptera frugiperda are incubated in the presence of IKKβ inhibitors in the wells of a Glutathione-Coated 96-Well Plate, which allows for substrate phosphorylation and capture in a single step. The phosphorylated GST-IκBα substrate is detected using an Anti-Phospho IκBα (Ser³²/Ser³⁶) 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 IKKβ activity.
      Materials provided• Glutathione Coated 96-well Plate (Kit Component No. JA9125): 1 plate supplied as 12 X 8-well strips
      • GST-IκBα (IKKβ Substrate) (Kit Component No. JA9127): 1 vial, 15 µg; please see label for lot-specific concentration
      • Anti-Phospho IκBα (Ser³²/Ser³⁶) (Kit Component No. JA9126): 1 vial, 15 µl
      • IKKβ, His·Tag®, Human, Recombinant, Spodoptera frugiperda (Kit Component No. 481404): 1 vial, 2.5 µg
      • Antibody Diluent (Kit Component No. JA7917): 1 bottle, 25 ml
      • Kinase Assay Buffer (Kit Component No. JA9130): 1 vial, 5 ml, supplied as a 5X solution
      • ATP/MgCl₂ Mix (Kit Component No. JA7914): 1 vial, 2 ml, supplied as a 5X solution
      • IKKβ Inhibitor, 500X (Kit Component No. JA9129): 1 vial, 50 µl IKK-2 Inhibitor IV (Cat. No. 401481): supplied as a 1 mM solution in DMSO
      • Kinase Stop Solution (Kit Component No. KP31517): 1 vial, 5 ml
      • ELISA 20X Plate Wash Concentrate (Kit Component No. JA1617): 1 bottle, 100 ml
      • HRP-Conjugate (Kit Component No. JA7643): 1 vial, 100 µl, supplied as a 400X solution
      • TMB Substrate (Kit Component No. JA1608): 1 bottle, 12 ml, ready to use
      • ELISA Stop Solution (Kit Component No. JA1616): 1 bottle, 12 ml, 2.5 N H₂SO₄ ready to use
      • Plate Sealers: 2 each
      Materials Required but not provided• Spectrophotometer capable of measuring absorbance in 96-well plates at 450 nm, preferably in the dual wavelength mode with a reference wavelength of 540-600 nm.
      Reagent preparationPrepare all reagents immediately prior to use. Following reconstitution of the IKKβ, His·Tag®, Human, Recombinant, Spodoptera frugiperda, aliquot and freeze at -70°C. The reconstituted enzyme will lose activity if subjected to more than 3 freeze/thaw cycles. The following table provides reagent preparation instructions to obtain the volume of Working Solutions (WS) required for one 8-well strip.

      Table 1: Reagent Preparation
      Detailed protocol1. Remove required number of strips from the Glutathione-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 4°C.
      2. Add the following components to designated wells in the specified order:

      Table 2: Addition of Assay Components

      *Recommendation for screening inhibitors: to estimate the inhibitory effect of investigational drugs or known kinase inhibitors we suggest to conduct experiments as outlined in the table below. Following the table, add the Assay Reagents to each well, mix thoroughly, and initiate the reaction by adding 10 µl of the ATP/MgCl2 Mix.


      Table 3: Assay Set-Up

      **Alternatively, the source of enzyme can be supplied by the end-user, but titration of the enzyme sample is recommended prior to inhibitor testing. ***Refers to unknown inhibitor diluted to an appropriate working concentration. If the IC50 of the inhibitor is not known titration of inhibitor is necessary. ****Additional optional control samples containing 10 µl of Inhibitor WS may also be included; the volume of H2O must be adjusted to 10 µl for these additional controls.


      3. Cover the plate with the Plate Sealer, mix the contents of the wells with a plate shaker or equivalent, and incubate for 30 min at 30°C.
      4. Stop the kinase reaction by adding 10 µl Kinase Stop Solution to each well. (Note: alternatively the reaction may also be stopped by discarding the contents of the well).
      5. Aspirate contents of the wells and wash the Glutathione-Coated 96-Well-Plate with ELISA Plate Wash 1X using 200 µl/well. Discard the ELISA Plate Wash 1X solution by shaking the contents into a sink and dry the wells by tapping the inverted plate on paper towels. Repeat for a total of 3 washes.
      6. Add 100 µl Anti-Phospho IκBα (Ser32/Ser36) WS 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 10-20 min at room temperature.
      11. Add 100 µl ELISA Stop Solution to each well and read the absorbance at 450 nm, preferably with a reference wavelength set at 540-600 nm.
      Assay characteristics and examples

      Figure 1: Enzyme Activity of Purified Recombinant IKKΒ

      Typical results obtained when using purified recombinant IKKΒ in the absence of inhibitors. The activity of IKKΒ, His·Tag®, Human, Recombinant, Spodoptera frugiperda (Cat. No. 481404) was measured as outlined in the Detailed Protocol. Please note that the specific activity of the enzyme may vary from lot to lot.


      Figure 2: Inhibition of IKKΒ Activity by IKKΒ Inhibitor IV

      IKKΒ, His·Tag®, Human, Recombinant, Spodoptera frugiperda (Cat. No. 481404) (5 ng; please note that the specific activity of the enzyme may vary from lot to lot) was incubated in the presence of increasing concentrations of IKK-2 Inhibitor IV (Cat. No. 401481) and 10 µM ATP and the activity was measured as outlined above in the Detailed Protocol.


      Figure 3: Inhibition of IKKΒ Activity by Staurosporine

      IKKΒ, His·Tag®, Human, Recombinant, Spodoptera frugiperda (Cat. No. 481404) (1.5 ng) was incubated in the presence of increasing concentrations of Staurosporine (Cat. No. 569397) and 10 µM ATP and the activity was measured as outlined above in the Detailed Protocol.
      Assay Range5-10 ng
      Registered TrademarksCalbiochem® and His·Tag® are registered trademarks of EMD Chemicals, Inc.
      K-LISA™ S·Tag™, and Interactive Pathways™ are trademarks of EMD Chemicals, Inc.