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CBA052 IP-Activity™ Bcr-Abl/c-Abl Kit

CBA052
  
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      Overview

      Replacement Information
      Description
      OverviewThis kit is intended as a tool to immunoprecipitate the Abl tyrosine kinase from a variety of samples including cell lysates and partially purified preparations. Immunoprecipitated Abl retains activity and can be used in activity and inhibitor assays as well as immunoblots.
      Catalogue NumberCBA052
      Brand Family Calbiochem®
      Materials Required but Not Delivered Cell lysis buffer (e.g. CytoBuster™ Protein Lysis Reagent, Cat. No. 71009 or PhosphoSafe™ Lysis Reagent, Cat. No. 71296) Phosphatase inhibitor cocktail (e.g. Phosphatase Inhibitor Cocktail Set I, Cat. No. 524624 or Phosphatase Inhibitor Cocktail Set II, Cat. No. 524625) Protease inhibitor cocktail (e.g. Protease Inhibitor Cocktail Set V, Cat. No. 539137 or Protease Inhibitor Cocktail Set VII, Cat. No. 539138) PTK activity kit (optional; e.g., K-LISA™ PTK Screening Kit, Cat. No. 539701) Western blotting equipment and reagents (optional)
      References
      ReferencesHantschel, O. and Superti-Furga, G. 2004. Nat. Rev. Mol. Cell Bio. 5, 33; Wang, J.Y. 1993. Curr. Opin. Gene. Dev. 3, 35; Lewis, J.M., et al. 1996. Proc. Natl. Acad. Sci. USA 93, 15174; Lewis, J.M. and Schwartz, M.A. 1998. J. Biol. Chem. 273, 14225; Renshaw, M.W., et al. 2000. Oncogene 19, 3216; Kain, K.H. and Klemke, R.L. 2001. J. Biol. Chem. 276, 16185.
      Product Information
      Anti-c-Abl (Ab-3) Mouse mAb (24-21), Protein G Agarose, 10X Wash Buffer, Staurosporine, and a user protocol.
      Applications
      Biological Information
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe Calbiochem® IP-Activity™ Bcr-Abl/c-Abl Kit is an immunoprecipitation kit that specifically immunoprecipitates Bcr-Abl and c-Abl from a variety of sources, including cell lysates and partially-purified preparations, using a mouse monoclonal anti-Abl antibody and Protein G Agarose. The Abl-protein-G complex retains kinase activity and can be used directly to evaluate Bcr-Abl and c-Abl activity. Used in conjunction with pan-kinase inhibitor, Staurosporine (included), the kit can be used to determine the relative IC₅₀ values of test inhibitors.
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage +2°C to +8°C
      Storage ConditionsUpon arrival store the Staurosporine at -20°C; store all other components at 4°C.
      Do not freeze Ok to freeze
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      Supplemental Information
      Specifications

      Documentation

      IP-Activity™ Bcr-Abl/c-Abl Kit Certificates of Analysis

      TitleLot Number
      CBA052

      References

      Reference overview
      Hantschel, O. and Superti-Furga, G. 2004. Nat. Rev. Mol. Cell Bio. 5, 33; Wang, J.Y. 1993. Curr. Opin. Gene. Dev. 3, 35; Lewis, J.M., et al. 1996. Proc. Natl. Acad. Sci. USA 93, 15174; Lewis, J.M. and Schwartz, M.A. 1998. J. Biol. Chem. 273, 14225; Renshaw, M.W., et al. 2000. Oncogene 19, 3216; Kain, K.H. and Klemke, R.L. 2001. J. Biol. Chem. 276, 16185.
      User Protocol

      Revision08-July-2008 JSW
      StorageUpon arrival store the Staurosporine at -20°C; store all other components at 4°C.
      Intended useThe Calbiochem® IP-Activity™ Bcr-Abl/c-Abl Kit is an immunoprecipitation kit that specifically immunoprecipitates Bcr-Abl and c-Abl from a variety of sources, including cell lysates and partially-purified preparations, using a mouse monoclonal anti-Abl antibody and Protein G Agarose. The Abl-protein-G complex retains kinase activity and can be used directly to evaluate Bcr-Abl and c-Abl activity. Used in conjunction with pan-kinase inhibitor, Staurosporine (included), the kit can be used to determine the relative IC₅₀ values of test inhibitors.
      BackgroundThe c-Abl proto-oncogene was isolated as the normal cellular counterpart of the Abelson murine leukemia virus oncogene, v-Abl. The Abl tyrosine kinase family is comprised of Abl and Arg (Abelson-related gene), which are nonreceptor tyrosine kinases that have been implicated in cellular responses resulting from growth factor stimulation, integrin activation, and various types of stress. The N-terminal region of c-Abl contains tandem Src homology 3 (SH3), SH2, and SH1 domains. In contrast to other nonreceptor tyrosine kinases, the C-terminus of c-Abl contains G- and F-actin binding domains (FABD), three nuclear localization sequences (NLS), one nuclear export sequence (NES), and three high-mobility-group-like boxes (HLB) that cooperatively bind to A/T-rich DNA regions. The human c-Abl protein is ubiquitously expressed as two 145 kDa isoforms (1a and 1b) as a result of alternate splicing of the first two exons. The 1a form is mostly cytoplasmic while the 1b form is myristoylated at the N-terminus and found predominantly in the nucleus. In contrast, the oncogenic Bcr-Abl and v-Abl proteins do not enter the nucleus. Moreover, in mammalian cells c-Abl exhibits different sub-cellular localization depending on the cell type. In fibroblasts c-Abl is found predominantly in the nucleus, while in primary haematopoietic cells and neurons it is largely found in the cytoplasm. The crystal structure of regulated Abl closely resembles that seen in structures of regulated Src-family kinases, but with unique differences. Binding of myristate to a hydrophobic pocket in the kinase domain induces a defined conformational change that enables intramolecular docking of the SH2 domain to the kinase domain, thereby enforcing an autoinhibited conformation. Nuclear c-Abl plays a role in transcription regulation, especially in response to DNA damage by ionizing radiation (IR) and DNA cross-linking agents, but not by UV radiation. In the nucleus, ataxia-telangiectasia-mutated protein (ATM) binds the SH3 domain of c-Abl and upon exposure to IR activates c-Abl by phosphorylating it at Ser465. Overexpression of c-Abl in fibroblasts leads to formation of a p53/c-Abl complex that stimulates p53 activity, resulting in G1 cell cycle arrest and apoptosis. In the cytoplasm, c-Abl is activated by growth factors and cell adhesion, localizing to regions of the cytoskeleton that include various F-actin structures such as focal adhesions, pseudopodia, lamellipodia, filopodia, membrane ruffles, and neuronal extensions and synapses. PDGF-dependent activation of c-Abl requires both Src family kinases and PLC-γ (PLC-γ1 in fibroblasts). Src phosphorylates c-Abl at Tyr412 in the activation loop, while PLC-γ is proposed to reduce PIP2 levels to activate c-Abl. Cell-adhesion-mediated activation is dependent on mechanisms that override the negative effect of F-actin. FABD keeps c-Abl in the inactive conformation in detached cells. Once activated c-Abl phosphorylates substrate proteins to regulate various F-actin-based processes, such as membrane ruffling, filopodial exploration, neurite extension and cell migration.
      Materials provided• Anti-c-Abl (Ab-3) Mouse mAb (24-21) (Kit Component No. JA9164): 1 vial, 250 µl supplied at 100 µg/ml in 50 mM sodium phosphate, 0.09% sodium azide, 0.2% gelatin, pH 7.0; MSDS available upon request • Protein G Agarose (Kit Component No. JA9165): 1 vial, 250 µl supplied as a 50% slurry in PBS, 20% ethanol, pH 7.0 • 10X Wash Buffer (Kit Component No. JA9167): 1 bottle, 15 ml, 10X TBS, 1% Tween®-20 detergent, pH 7.0 • Staurosporine (Kit Component No. JA9166): 1 vial, 20 µl, 1 mM in DMSO; protect from light
      Materials Required but not provided Cell lysis buffer (e.g. CytoBuster™ Protein Lysis Reagent, Cat. No. 71009 or PhosphoSafe™ Lysis Reagent, Cat. No. 71296) Phosphatase inhibitor cocktail (e.g. Phosphatase Inhibitor Cocktail Set I, Cat. No. 524624 or Phosphatase Inhibitor Cocktail Set II, Cat. No. 524625) Protease inhibitor cocktail (e.g. Protease Inhibitor Cocktail Set V, Cat. No. 539137 or Protease Inhibitor Cocktail Set VII, Cat. No. 539138) PTK activity kit (optional; e.g., K-LISA™ PTK Screening Kit, Cat. No. 539701) Western blotting equipment and reagents (optional)
      Reagent preparation 1X Wash Buffer: prepare 1X Wash Buffer by adding 1 part 10X Wash Buffer to 9 parts ultrapure H2O.
      Detailed protocol1. Prepare cell lysate from monolayer cells, suspension cells, or cell pellets using 0.5 ml PhosphoSafe™ Lysis Reagent (or equivalent cell lysis buffer containing phosphatase inhibitors) per 1 x 107 cells and proceed with the lysis per the manufacturer's user protocol. 2. Centrifuge for 5 min at 16,000 x g at 4°C to pellet the insoluble cellular debris. Transfer the supernatant to a clean tube. 3. Pre-clear lysate by adding 25 µl Protein G-Agarose per ml cell lysate. Place the tube on a rocker and mix (end-to-end) for 1 h at 4°C. 4. Centrifuge for 5 min at 16,000 x g at 4°C to pellet the Protein G-Agarose. Transfer the supernatant to a clean tube. 5. Add 2.5 µg Anti-Abl mouse mAb per ml cell lysate and mix on a rocker (end-to-end) overnight at 4°C. 6. Add 25 µl Protein G-Agarose per ml cell lysate and mix on a rocker (end-to-end) for 1 h at 4°C. 7. Centrifuge for 5 min at 16,000 x g at 4°C to pellet the Abl-antibody-Protein G-Agarose complex. Discard the supernatant. 8. Add 1 ml 1X Wash Buffer, invert several times to mix, and centrifuge for 5 min at 16,000 x g at 4°C to pellet the complex; remove the supernatant and discard. Repeat for a total of 5 washes. 9. Resuspend pellet in appropriate buffer for further assay. Appropriate buffers include assay buffer (e.g., ± inhibitor) for activity assays or SDS-PAGE sample buffer for immunoblot analysis.
      Assay characteristics and examplesK-562 cells (for Bcr-Abl) were grown in RPMI with 10% FBS and 2 mM L-glutamine. NIH3T3 cells (c-Abl knockout) were transfected with empty vector (Abl⁻) or vector containing c-Abl (Abl+) and grown in DMEM with 10% FBS and 2 mM L-glutamine. Cell lysates were prepared using PhosphoSafe™ Lysis Reagent and immunoprecipitated as indicated in the Detailed Protocol above. Activities were measured using the K-LISA™ PTK Screening Kit recommended protocol and a K-LISA™ PTK EAY Reaction Plate. For staurosporine inhibition studies (Figure 6), the inhibitor was pre-incubated with immunoprecipitated c-Abl in reaction buffer for 15 min at room temperature before addition of ATP to initiate the activity assay.

      Figure 1: Sample Blot

      Pre-cleared Lysate from K-562 Cells (lane 1) and Bcr-Abl Immunoprecipitated using Anti-Abl mouse mAb (lane 2, arrow).

      Figure 2: Titration of Bcr-Abl Activity

      Bcr-Abl was immunoprecipitated from K-562 cell lysate using 2.5 µg Anti-Abl mouse mAb per ml of cell lysate. Triplicate data were averaged, corrected for reaction buffer only, and absorbance at 595 nm subtracted for plate background. Dilutions were prepared reaction buffer and assumed a 25 µl pellet volume.

      Figure 3: Sample Blot

      NIH3T3 Cells (c-Abl knockout) Transfected with Empty Vector (Abl-) or Vector Containing c-Abl (Abl+). Lanes 1 and 3: pre-cleared lysates; Lanes 2 and 4: immunoprecipitates using Anti-Abl mouse mAb.

      Figure 4: c-Abl Activity

      c-Abl was immunoprecipitated using 2.5 µg Anti-Abl mouse mAb per ml of cell lysate. Triplicate data were averaged, corrected for reaction buffer only, and absorbance at 595 nm subtracted for plate background. S/N ~11.5.

      Figure 5: Titration of c-Abl Activity

      c-Abl was immunoprecipitated from Abl+ cell lysates using 2.5 µg Anti-Abl mouse mAb per ml of cell lysate. Triplicate data were averaged, corrected for reaction buffer only, and absorbance at 595 nm subtracted for plate background. Dilutions were prepared in reaction buffer and assumed a 25 µl pellet volume.

      Figure 6: Staurosporine Inhibition of c-Abl Activity

      c-Abl was immunoprecipitated from Abl+ cell lysates using 2.5 µg Anti-Abl mouse mAb per ml of cell lysate. Triplicate data were averaged, corrected for reaction buffer only, and absorbance at 595 nm subtracted for plate background. Dilution was 160-fold into reaction buffer and assumed a 25 µl pellet volume.

      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc. Tween® is a registered trademark of ICI Americas, Inc. Interactive Pathways™, K-LISA™, CytoBuster™, and PhosphoSafe™ are trademarks of EMD Chemicals, Inc.