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CBA056 IP-Activity™ Src Kit

IP-Activity™ Src Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
CBA056
  
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      Overview

      Replacement Information
      Description
      OverviewThis kit immunoprecipitates c-Src and v-Src tyrosine kinase from a variety of sources, including cell lysates and partially-purified preparations. The Src/Protein G complex retains kinase activity and can be used directly to evaluate Src activity.
      Catalogue NumberCBA056
      Brand Family Calbiochem®
      Materials Required but Not Delivered• Cell lysis buffer (e.g. CytoBuster™ Protein Extraction Reagent, Cat. No. 71009 or PhosphoSafe™ Extraction 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) • Immunoblotting equipment and reagents (optional)
      References
      ReferencesBoggon, T.J. and Eck, M.J. 2004. Oncogene 23, 7918. Bromann, P.A., et al. 2004. Oncogene 23, 7957. Ishizawar, R. and Parsons, S.J. 2004 Cancer Cell 6, 209. Martin, G.S. 2004. Oncogene 23, 7910. Parsons, S.J. and Parsons, J.T. 2004. Oncogene 23, 7906. Biscardi, J.S., et al. 2000. Breast Cancer Res. 2, 203. Hunter, T. and Sefton, B.M. 1980. Proc. Natl. Acad. Sci. USA 77, 1311.
      Product Information
      Kit containsAnti-v-Src Mouse mAb (327), Protein G Agarose, 10X Wash Buffer, Staurosporine and a user protocol
      Positive controlA431 Cells
      Applications
      Biological Information
      Sample TypeCell lysates
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe Calbiochem® IP-Activity™ Src Kit is an immunoprecipitation kit that specifically immunoprecipitates c-Src and v-Src from a variety of sources, including cell lysates and partially-purified preparations, using a mouse monoclonal anti-Src antibody and Protein G Agarose. The Src-protein-G complex retains kinase activity and can be used directly to evaluate Src 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 Multiple storage requirements
      Storage ConditionsUpon arrival store the Staurosporine at -20°C; store all other components at 4°C.
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsAnti-v-Src Mouse mAb (327), Protein G Agarose, 10X Wash Buffer, Staurosporine and a user protocol
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      CBA056 0

      Documentation

      IP-Activity™ Src Kit Certificates of Analysis

      TitleLot Number
      CBA056

      References

      Reference overview
      Boggon, T.J. and Eck, M.J. 2004. Oncogene 23, 7918. Bromann, P.A., et al. 2004. Oncogene 23, 7957. Ishizawar, R. and Parsons, S.J. 2004 Cancer Cell 6, 209. Martin, G.S. 2004. Oncogene 23, 7910. Parsons, S.J. and Parsons, J.T. 2004. Oncogene 23, 7906. Biscardi, J.S., et al. 2000. Breast Cancer Res. 2, 203. Hunter, T. and Sefton, B.M. 1980. Proc. Natl. Acad. Sci. USA 77, 1311.
      User Protocol

      Revision02-January-2008 JSW
      StorageUpon arrival store the Staurosporine at -20°C; store all other components at 4°C.
      Intended useThe Calbiochem® IP-Activity™ Src Kit is an immunoprecipitation kit that specifically immunoprecipitates c-Src and v-Src from a variety of sources, including cell lysates and partially-purified preparations, using a mouse monoclonal anti-Src antibody and Protein G Agarose. The Src-protein-G complex retains kinase activity and can be used directly to evaluate Src 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 history of Src is the history of the discovery of protein tyrosine kinases. Almost a century ago, Peyton Rous discovered that injections of cell-free extracts from chicken tumors could cause tumors in naive chickens (i.e., viral transmission). In the 1950's, some strains of the Rous sarcoma virus (RSV) were found to induce tumors in mammalian hosts and in 1960 it was discovered that the virus contained a single-stranded RNA genome. In 1977 a 60 kDa phosphoprotein was immunoprecipitated from RSV-transformed cells from the sera of tumor-bearing rabbits. A year later it was demonstrated that the protein was a kinase (v-Src). Although initially described as a threonine-specific kinase, in 1979 Tony Hunter's group showed that v-Src activity was that of a protein tyrosine kinase and in the process opened the door to protein tyrosine kinases. This was rapidly followed by the discovery that v-Abl and EGFR were also members of the protein tyrosine kinase family. It has been a little over a quarter of a century since the discovery of the proto-oncogene c-Src. Significantly, v-Src has not been found to be an etiological agent in human cancers, while originally c-Src was found to be rarely mutated in human cancers and when over-expressed in normal cells appeared non- or weakly oncogenic. However, recent studies show that c-Src exhibits elevated levels and activity in numerous types of human cancer. It is a critical protein in multiple signaling pathways involved in proliferation, survival, metastasis, and angiogenesis. The Src family is composed of nine members (Src, Lck, Hck, Fyn, Blk, Lyn, Fgr, Yes and Yrk). All members share a conserved domain structure consisting of consecutive SH4, SH3, SH2, and SH1 domains. The SH4 domain is a membrane-targeting region at the N-terminus, which is always myristoylated (sometimes palmitoylated) and followed by a unique (50-70 aa) region that is divergent among the family members. Src contains a Class 1 SH3 domain (~62 aa) that recognizes a proline-rich peptide motif (RKXXPXXP), which forms a left-handed polyproline type II helix. The Src SH2 domain (~ 98 aa) binds to specific phosphotyrosine (pTyr)-containing peptide motifs (preferentially, pYEEI motif). Activation of the SH1 or kinase domain (~254 aa) requires autophosphorylation at Tyr418. Finally, c-Src contains a short C-terminal negative regulatory region that is absent in v-Src. Inactivation by Csk (pTyr529 in c-Src) promotes the assembly of the SH2, SH3, and SH4 domains into an autoinhibited conformation. Examples of the importance of Src in human cancer can be seen through its interactions with the EGF receptor family, focal adhesion kinase (FAK), and steroid hormone receptors. In breast cancer, c-Src and members of the EGFR family are over-expressed in ~70% of tumors and c-Src activity is necessary for HER2-mediated anchorage-independent growth, motility, and survival. In EGFR, c-Src activates the RTK at Tyr845, which then activates at least two distinct signaling pathways via Stat5b (EGF-induced cell proliferation) and through Cox II (enhancement of cell survival). In addition, c-Src phosphorylation of clathrin and dynamin (involved in the internalization of EGFR) enhances the endosomal pool of activated receptors that continue to transduce signals until degraded. c-Src also facilitates the ubiquitination and proteasomal degradation of Cbl, which reduces down-regulation of EGFR. The association of cell surface molecules with the N-terminus of FAK leads to autophosphorylation of FAK at Tyr397. Consequently, the c-Src-SH2 group binds to FAK at pTyr397 resulting in c-Src activation and phosphorylation of FAK at Tyr576, Tyr577, Tyr861, and Tyr925 followed by the generation of docking sites for Grb2 and other signaling proteins. The FAK/c-Src complex also phosphorylates cytoskeletal adapter proteins, paxillin and Cas. These events recruit and activate regulators of ERK, JNK, and Rho signaling pathways, which modulate gene expression of transcription factors and target proteins involved in cell motility and invasion. Finally, it has been found that the steroid hormones estrogen, progesterone, and androgen can stimulate the c-Src/p21Ras/ERK pathway. Studies have shown that activated c-Src can phosphorylate the ER and that PR can react directly with c-Src, but the details have not been fully elucidated.
      Materials provided• Anti-v-Src Mouse mAb (327) (Kit Component No. JA9193): 1 vial, 250 µl supplied at 100 µg/ml in 50 mM sodium phosphate, 0.09% sodium azide, 0.2% gelatin, pH 7.0 • 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 Extraction Reagent, Cat. No. 71009 or PhosphoSafe™ Extraction 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) • Immunoblotting equipment and reagents (optional)
      Precautions and recommendations• To preserve the phosphorylation state it is recommended that a cocktail of phosphatase inhibitor(s) be included in the cell lysis buffer • Always work on ice • To prevent high background it is recommended that cell lysates be pre-cleared with the Protein G Agarose prior to immunoprecipitation with Anti-v-Src Mouse mAb (327) • The Src-antibody-protein-G-agarose complex can be resuspended in the K-LISA™ reaction buffer and used directly in the K-LISA™ PTK Screening Kit (Cat. No. 539701)
      Reagent preparation1X 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™ Extraction Reagent (or equivalent cell lysis buffer containing phosphatase inhibitors) per 1 x 107 cells and proceed with the extraction 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-v-Src Mouse mAb (327) 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 Src-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 examplesTo obtain the following data, A431 cells were grown in DMEM with 10% FBS and 2 mM L-glutamine. Cell lysates were prepared using PhosphoSafe™ Extraction Reagent (Cat. No. 71296) and immunoprecipitated as indicated in the Detailed Protocol above. Activities were measured using the K-LISA™ PTK Screening Kit (Cat. No. 539701) according to the recommended protocol and a K-LISA™ PTK EY Reaction Plate (Cat. No. 539704). For staurosporine inhibition studies (Figure 3), the inhibitor was pre-incubated with immunoprecipitated c-Src in reaction buffer for 15 min at room temperature before addition of ATP to initiate the activity assay.

      Figure 1: Immunoprecipitation of c-Src from A431 Cell Lysates

      Lane 1: Pre-cleared lysate; Lane 2 : Immunoprecipitate using Anti-v-Src Mouse mAb (327).










      Figure 2: Titration of c-Src Activity

      c-Src was immuno-precipitated from A431 cell lysates using 2.5 µg Anti-v-Src Mouse mAb (327) per ml 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 3: Staurosporine Inhibition of c-Src Activity

      Src was immunoprecipitated from A431 cell lysates using 2.5 µg Anti-v-Src Mouse mAb (327) per ml 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.

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