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CBA020 K-LISA™ Checkpoint Activity Kit

K-LISA™ Checkpoint Activity Kit: Ficha de datos de seguridad (MSDS o SDS), certificado de análisis y de calidad (CoA y CoQ), expedientes, folletos y otros documentos disponibles.

Sinónimos: K-LISA™ Chk1/Chk2 Activity Kit

CBA020
  
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      Descripción

      Replacement Information

      Tabla espec. clave

      Detection Methods
      Colorimetric
      Description
      Overview

      This product has been discontinued.





      The K-LISA™ Checkpoint Activity Kit is a rapid, sensitive, ELISA-based activity assay suitable for measuring the kinase activity of purified or partially purified Chk1 and Chk2 preparations, in vitro Chk1 and Chk2 inhibitor screening, and for assessing the regulation of Chk1 and Chk2 in cell signaling. The assay utilizes a biotinylated peptide substrate (KKKVSRSGLYRSPSMPENLNRPR) that is phosphorylated on the third serine by Chk1 and Chk2. Biotinylated Chk substrate and sample containing Chk1 or Chk2 are incubated in the presence of ATP in wells of the streptavidin-coated 96-well plate, which allows for phosphorylation and substrate capture in a single step.

      • Highly versatile: can detect kinase activity in purified or partially purified Chk1 and Chk2 preparations.
      • Highly sensitive: can detect kinase activity at picogram (Chk1) and nanogram levels (Chk2).
      • Highly convenient: 96-well format, non-radioactive detection.
      • Highly useful: enables high throughput screening of kinase inhibitors.

      Catalogue NumberCBA020
      Brand Family Calbiochem®
      SynonymsK-LISA™ Chk1/Chk2 Activity Kit
      Application Data
      The activity of His•Tag®, Human, Recombinant Chk1 (Cat. No. 220479) (17-2200 pg) was determined using protocol A as indicated in the Detailed Protocol section. Assay range: 34 pg-1100 pg (740 Units/mg).

      The activity of Human, Recombinant Chk2 (0.06-60 ng) was determined using protocol A in the Detailed Protocol section. Assay range: 200 pg-20 ng (1283 Units/mg).
      Materials Required but Not Delivered• Chk1 or Chk2-containing sample
      • Anti-Chk1, Human (Sheep) (for immunoprecipitating Chk1) Cat. No. PC423
      • Anti-Chk2, Human (Rabbit) (for immunoprecipitating Chk2) Cat. No. DR1036
      • Protein A Agarose Suspension (Cat. No. IP02), Protein G-Plus Agarose Suspension (Cat. No. IP04), or Protein A/Protein G-Plus Agarose Suspension (Cat. No. IP05) (for immunoprecipitation of Chk1 and Chk2)
      • 1X PBS
      • RIPA Buffer (Tris-HCl: 50 mM, pH 7.4, NP-40: 1%, Na-deoxycholate: 0.25% NaCl: 150 mM, EDTA: 1 mM)
      • PhosphoSafe™ Extraction Buffer (Cat. No. 71296) or equivalent for preparation of cell lysates
      • Wash bottle or multichannel dispenser for washing
      • Spectrophotometer capable of measuring absorbance in 96-well plates at 450nm, preferably in the dual wavelength mode at 450nm against a reference wavelength of 540 or 595 nm.
      References
      ReferencesBartek, J., and Lukas, J. 2003. Cancer Cell 3, 421.
      O'Neill T., et al. 2002. J Biol. Chem. 277, 16102.
      Jackson, J.R., et al. 2000. Cancer Res. 60, 566.
      Liu, Q., et al. 2000. Genes Dev. 14, 1448.
      Product Information
      Detection methodColorimetric
      Form96 Tests
      Format96-well plate
      Kit containsBiotinylated Chk1 Substrate, Phosphoserine Detector Antibody, Streptavidin Coated 96-Well Plate Strips, HRP-Conjugated Secondary Antibody, Serine/Threonine Kinase Assay Buffer, ATP/MgCl₂, Chk1 Inhibitor, Wash Buffer, TMB Substrate, Stop Solution, Plate Sealers, and a user protocol. Purified Chk1 is available separately (Cat. No. 220479).
      Applications
      Biological Information
      Assay range25-725 µU (Chk1) and 260 µU - 26 mU (Chk2)
      Assay time3 h
      Sample TypeCell lysates, tissue extracts, purified enzymes
      Physicochemical Information
      Sensitivity25 pg (0.018 mU) purified Chk1
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe K-LISA™ Checkpoint Activity Kit is suitable for measuring the kinase activity of purified or partially purified Chk1 and Chk2 preparations, in vitro Chk1 and Chk2 inhibitor screening, and for assessing the regulation of Chk1 and Chk2 in cell signaling.
      Storage and Shipping Information
      Ship Code Dry Ice Only
      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 -20°C
      Storage ConditionsUpon arrival store the Streptavidin-Coated 96-Well Plate at 4°C and the remaining components of the kit at -20°C.
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsBiotinylated Chk1 Substrate, Phosphoserine Detector Antibody, Streptavidin Coated 96-Well Plate Strips, HRP-Conjugated Secondary Antibody, Serine/Threonine Kinase Assay Buffer, ATP/MgCl₂, Chk1 Inhibitor, Wash Buffer, TMB Substrate, Stop Solution, Plate Sealers, and a user protocol. Purified Chk1 is available separately (Cat. No. 220479).
      Specifications
      Global Trade Item Number
      Número de referencia GTIN
      CBA020 0

      Documentation

      K-LISA™ Checkpoint Activity Kit Certificados de análisis

      CargoNúmero de lote
      CBA020

      Referencias bibliográficas

      Visión general referencias
      Bartek, J., and Lukas, J. 2003. Cancer Cell 3, 421.
      O'Neill T., et al. 2002. J Biol. Chem. 277, 16102.
      Jackson, J.R., et al. 2000. Cancer Res. 60, 566.
      Liu, Q., et al. 2000. Genes Dev. 14, 1448.

      Folleto

      Cargo
      Kit SourceBook - 2nd Edition EURO
      Kits SourceBook - 2nd Edition GBP
      Protein Kinase Assay and Detection Kits Brochure
      Protocolo de usuario

      Revision20-February-2009 JSW
      SynonymsK-LISA™ Chk1/Chk2 Activity Kit
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      StorageUpon arrival store the Streptavidin-Coated 96-Well Plate at 4°C and the remaining components of the kit at -20°C.
      Intended useThe K-LISA™ Checkpoint Activity Kit is suitable for measuring the kinase activity of purified or partially purified Chk1 and Chk2 preparations, in vitro Chk1 and Chk2 inhibitor screening, and for assessing the regulation of Chk1 and Chk2 in cell signaling.
      BackgroundIn mammalian cells the checkpoint response to DNA damage or replication stress regulates cellular processes such as cell-cycle progression, apoptosis, and DNA replication. Checkpoint kinase (Chk1 and Chk2) activity is required for the normal G2 checkpoint response to diverse cellular stresses, in particular those that result in DNA damage such as ionizing radiation (IR), UV radiation, and topoisomerase inhibitors. In response to DNA damage Chk1 is activated by phosphorylation of Ser345 by ATR and subsequently phosphorylates CDC25C, an activator of Cdk2/Cyclin B. Phosphorylation of CDC25C by Chk1 interferes with 14-3-3 protein binding and restricts CDC25C to the cytoplasm, resulting in G2 arrest. Similarly, Chk1 is necessary for sensing replication stress induced by DNA synthesis inhibitors. For example, the DNAa polymerase inhibitor, aphidicholin induces an S phase checkpoint that also requires ATR-dependent Chk1 activity. Known cellular targets of Chk1 include p53, CDC25A, CDC25B, CDC25C, and CK2β. Unlike Chk1, Chk2 is not necessary for viability. Nevertheless, Chk2 signals double strand breaks (DSBs) via ATM activation and phosphorylates some of the same substrates as Chk1. Whereas Chk2 is mainly activated in response to DSBs, Chk1 is activated in response to replication stress. In contrast to Chk1 activity that is detected constitutively in cycling cells, inducible Chk2 activity is detectable in cells exposed to genotoxic stress. Cancer cells often adapt to DNA damaging chemotherapeutic agents as a means of escaping apoptosis. This adaptive mechanism may include cell cycle arrest and repair of damaged DNA. The checkpoint proteins are important in oncogenesis and, thus, a potential target for cancer therapy. Inhibitors of Chk1 and Chk2 include the general kinase inhibitor, staurosporine, while UCN-01 and SB218078 are specific inhibitors of Chk1.
      Principles of the assayThe K-LISA™ Checkpoint Activity Kit is an ELISA-based activity assay that utilizes a biotinylated peptide substrate (KKKVSRSGLYRSPSMPENLNRPR) that is phosphorylated on the fourth serine (bold face) by Chk1and Chk2. Biotinylated Chk Substrate and sample containing Chk1 or Chk2 are incubated in the presence of ATP in wells of the Streptavidin-Coated 96-Well Plate, which allows for phosphorylation and substrate capture in a single step. The phosphorylated substrate is detected with the Phosphoserine Detection Antibody, followed by the HRP-Antibody Conjugate and color development with TMB Substrate. Sensitivity is increased by the addition of ELISA Stop Solution, and relative activity is determined by reading the absorbance at dual wavelengths 450/540 nm or 450/595 nm. Recommended sample types include Chk1 or Chk2 that has been immunoprecipitated from crude cell lysates or purified Chk1 or Chk2 proteins. Addition of Inhibitor (staurosporine) serves as a negative control. Inhibition profiles can be generated based on Chk activity in the presence and absence of test inhibitor(s).
      Materials provided• Streptavidin-Coated 96-Well Plate (Kit Componet No. JA7912-1EA): 1 each, 12 x 8-well strips, pre-blocked
      • Substrate, Biotinylated (Kit Componet No. JA7920-10UG): 1 vial, 10 µg
      • *Phosphoserine Detection Antibody (Kit Componet No. JA7911-15UL): 1 vial, 15 µl
      • Kinase Assay Buffer (5X) (Kit Componet No. JA7913-5ML): 1 vial, 5 ml
      • 5X ATP/MgCl2 Mix (Kit Componet No. JA7914-2ML): 1 vial, 2 ml
      • 100X Inhibitor (Kit Componet No. JA7915-25UL): 1 vial, 25 µl staurosporine in DMSO
      • Kinase Stop Solution (Kit Componet No. KP31517-25ML): 1 vial, 5 ml
      • ELISA 20X Plate Wash Concentrate (Kit Componet No. JA1617-5ML): 1 bottle, 100 ml
      • Antibody Diluent (Kit Componet No. JA7917-100ML): 1 bottle, 25 ml
      • HRP-Antibody Conjugate (Kit Componet No. JA7922-100UL): 1 bottle, 100 ml, supplied as a 400X solution
      • TMB substrate (Kit Componet No. JA1608-12ML): 1 bottle, 12 ml, ready-to-use
      • ELISA Stop Solution (Kit Componet No. JA1616-12ML): 1 bottle, 12 ml, 2.5N H₂SO₄
      • Plate Sealers (Kit Componet No. JB155-EA): 2 each
      Materials Required but not provided• Chk1 or Chk2-containing sample
      • Anti-Chk1, Human (Sheep) (for immunoprecipitating Chk1) Cat. No. PC423
      • Anti-Chk2, Human (Rabbit) (for immunoprecipitating Chk2) Cat. No. DR1036
      • Protein A Agarose Suspension (Cat. No. IP02), Protein G-Plus Agarose Suspension (Cat. No. IP04), or Protein A/Protein G-Plus Agarose Suspension (Cat. No. IP05) (for immunoprecipitation of Chk1 and Chk2)
      • 1X PBS
      • RIPA Buffer (Tris-HCl: 50 mM, pH 7.4, NP-40: 1%, Na-deoxycholate: 0.25% NaCl: 150 mM, EDTA: 1 mM)
      • PhosphoSafe™ Extraction Buffer (Cat. No. 71296) or equivalent for preparation of cell lysates
      • Wash bottle or multichannel dispenser for washing
      • Spectrophotometer capable of measuring absorbance in 96-well plates at 450nm, preferably in the dual wavelength mode at 450nm against a reference wavelength of 540 or 595 nm.
      Reagent preparation

      Table 1: Preparation of Reagents

      Prepare all reagents immediately prior to use. The above table provides reagent preparation instructions to obtain the Volume of Working Solutions (WS) required for one 8-well strip.
      Detailed protocolA. Checkpoint Kinase Activity and Inhibitor Screening Assay: recommended for purified or partially purified Chk1 or Chk2 preparations and for inhibitor screening.

      1. Remove required number of strips from the Streptavidin-Coated 96-Well Plate and place them in the 96-well frame. Return the unused strips to the foil pouch and reseal the entire edge of the pouch with tape. Store the remaining strips at 4°C.
      2. Add the following components to each well in the specified order:

      Table 2: Order of Reagent Addition Protocol A

      *For inhibitor screening assay; if no inhibitor is to be included, up to 20 µl Chk Sample can be added or inhibitor can be substituted with 10 µl dH2O.


      3. Cover the plate with the Plate Sealer, mix with a plate shaker or equivalent for 30 s, and incubate for 30 min at 30°C.
      4. Stop the kinase reaction by adding 10 µl Kinase Stop Solution to each well. (Reaction may also be stopped simply by discarding the contents of the wells).
      5. Aspirate contents of the wells and wash the Streptavidin-Coated 96-Well Plate 3 - 4 times with 1X ELISA Plate Wash (200 µl per well). Shake out the wash solution and dry the wells by tapping the inverted plate on paper towels.
      6. Add 100 µl Phosphoserine Detection Antibody WS to each well and incubate 1 h at room temperature.
      7. Wash the plate as in step 5.
      8. Add 100 µl HRP-Antibody Conjugate WS to each well and incubate 1 h at room temperature.
      9. Wash the plate as in step 5.
      10. Add 100 µl TMB Substrate to each well and incubate 5-20 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 540-595 nm.

      B. Assay for Activity of Immunoprecipitated Chk1 or Chk2: recommended for analysis of checkpoint kinase activity in samples immunoprecipitated from cell lysates. Note: K-LISA™ Checkpoint Activity Kit does not include an antibody for immunoprecipitation of active Chk1 or Chk2 from cell lysates. It is important is to use an antibody that will immunoprecipitate the enzyme without affecting the catalytic activity of the kinase and is capable of immunoprecipitating Chk1 or Chk2 efficiently from crude biological samples.
      1. Prepare cell lysates using PhosphoSafe™ Lysis Buffer (Cat. No. 71296) or other cell lysis buffer of choice. Pre-clear 0.5 ml cell lysate (total protein concentration = 2-5 mg/ml) by adding 15 µl Protein G-Plus, Protein A, or Protein A/Protein G-Plus agarose beads (settled bed volume) and incubating 15 min at 4°C.
      2. Centrifuge at 4000 rpm for 5 min at 4°C in a microcentrifuge to pellet the agarose beads. Transfer the pre-cleared lysate to a fresh tube.
      3. Add 5-10 µl appropriate Chk antibody (Cat. No. PC423 for Chk1 or Cat. No. DR1036 for Chk2) to the pre-cleared lysate and rotate 1 h at 4°C.
      4. Add 50 µl Protein G-Plus, Protein A, or Protein A/Protein G-Plus agarose beads (settled bed volume) and rotate 60-90 min at 4°C.
      5. Centrifuge at 4000 rpm for 5 min at 4°C in a microcentrifuge to pellet the agarose beads (now bound to immunoprecipitated Chk protein). Carefully remove the supernatant with a pipet tip and discard.
      6. Wash the agarose beads with 0.5 ml RIPA buffer, centrifuge at 4000 rpm for 5 min at +4°C to pellet the agarose beads, and discard the supernatant. Repeat for a total of 3 washes. Wash the pellet one time in 1X Kinase Assay Buffer. Carefully remove excess liquid following the final wash so as not to disturb the loose pellet of agarose beads.
      7. Add the following components in the specified order to the agarose beads in each tube, gently mix to evenly suspend the beads, and incubate at 30°C for 30 min.

      Table 3: Order of Reagent Addition Protocol B


      8. Add 10 µl Kinase Stop Solution to each tube, mix briefly by gently tapping the tube. DO NOT VORTEX.
      9. Centrifuge at 4000 rpm for 5 min at 4°C to pellet the agarose beads. Transfer supernatant to a fresh tube. The supernatant should be used immediately or stored at -70°C until the assay can be completed.
      10. Remove required number of strips from the Streptavidin-Coated 96-Well Plate and place them in a 96-well frame. Return the unused strips to the foil pouch and seal the entire edge of the pouch with tape. Store the remaining strips at 4°C.
      11. Add 10-50 µl supernatant from step 9 to designated wells. If less than 50 µl is used, add 1X PBS to a final volume of 50 µl. Incubate for 30-60 min at 30°C.
      12. Aspirate the contents of each well and wash the Streptavidin-Coated 96-Well Plate 3-4 times with 1X ELISA Plate Wash. Dry the wells by tapping the inverted plate on paper towels.
      13. Add 100 µl Phosphoserine Detection Antibody WS to each well and incubate 1 h at room temperature.
      14. Wash the plate as in step 12.
      15. Add 100 µl HRP-Conjugate WS to each well and incubate 1 h at room temperature.
      16. Wash the plate as in step 12.
      17. Add 100 µl TMB Substrate to each well and incubate 5-20 min. at room temperature.
      18. Add 100 µl ELISA Stop Solution to each well and read the absorbance at 450 nm, preferably with a reference wavelength set at 540-595 nm.
      Assay characteristics and examples

      Figure 1: Activity of Purified Chk1

      The activity of His•Tag®, Human, Recombinant Chk1 (Cat. No. 220479) (17-2200 pg) was determined using protocol A as indicated in the Detailed Protocol section. Assay range: 34 pg-1100 pg (740 Units/mg).


      Figure 2: Staurosporine Inhibition of Chk1

      The activity of His•Tag®, Human, Recombinant (Cat. No. 220479) (1.1 ng) was determined as in Figure 1 in the presence of increasing Staurosporine concentration.


      Figure 3: Activity of Chk1 Immunoprecipitated From HeLa Cell Lysate

      Cell lysate was prepared using 0.5 ml PhosphoSafe™ Lysis Buffer (Cat. No. 71296) for each 10 cm cell culture dish, according to the standard protocol. Cell lysates were either used immediately or stored at -70°C until use. Equal amounts of total protein (500 µg) were immunoprecipitated using the indicated antibody and activity was determined according to Detailed Protocol B.


      Figure 4: Assay Characteristics and Examples

      Inhibition of immunoprecipitate-associated Chk1 kinase activity from HeLa cell extracts and Chk1, His•Tag®, Human, Recombinant (1.1 ng) using the Chk1-specific inhibitor, SB218078, and staurosporine, respectively. Immunoprecipitation of Chk1 was performed as in Figure 3. Kinase assays were performed either in the presence or absence of 10 nM staurosporine for purified Chk1 as in Figure 2 or 100 nM of SB218078 (Cat. No. 559402) for immunoprecipitated Chk1.


      Figure 5: Activity of Purified Chk2

      The activity of Human, Recombinant Chk2 (0.06-60 ng) was determined using protocol A in the Detailed Protocol section. Assay range: 200 pg-20 ng (1283 Units/mg).


      Figure 6: Staurosporine Inhibition of Chk2

      The activity of Human, Recombinant Chk2 (6 ng) was determined using protocol A in the Detailed Protocol section in the presence of increasing Staurosporine concentration.


      Figure 7: Activity of Chk2 Immunoprecipitated from HeLa Cell Lysate

      Near-confluent HeLa cells were treated with UV (50 J/m2) or camptothecin (10 µM) for 2 h. Following exposure to UV the cells were incubated in fresh medium for 2 h at 37°C in a CO2 incubator. Immunoprecipitation of Chk2 was performed using the indicated antibody and kinase activity was determined according to Detailed Protocol B.
      Sensitivity25 pg (0.018 mU) purified Chk1
      Assay Range25-725 µU (Chk1) and 260 µU - 26 mU (Chk2)
      Registered TrademarksCalbiochem® and His•Tag® are registered trademarks of EMD Chemicals, Inc.
      K-LISA™,InteractivePathways™, and PhosphoSafe™ are trademarks of EMD Chemicals, Inc.
      Manufactured by Cell Signaling Technology®, a registered trademark of Cell Signaling Technology, Inc.
      *Sold under license of U.S. patent 6,441,140