Revision 1

#76696Store at -20C

1 Kit

(7 x 20 microliters)

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

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3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
DNA-PKcs (E6U3A) Rabbit mAb 38168 20 µl 450 kDa Rabbit IgG
Phospho-DNA-PKcs (Ser2056) (E9J4G) Rabbit mAb 68716 20 µl 450 kDa Rabbit IgG
Ku70 (D10A7) Rabbit mAb 4588 20 µl 70 kDa Rabbit IgG
Ku80 (C48E7) Rabbit mAb 2180 20 µl 86 kDa Rabbit IgG
DNA Ligase IV (D5N5N) Rabbit mAb 14649 20 µl 100 kDa Rabbit IgG
XLF Antibody 2854 20 µl 39 kDa Rabbit 
Artemis (D7O8V) Rabbit mAb 13381 20 µl 90 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

Description

The Non-Homologous End Joining (NHEJ) DNA Repair Antibody Sampler Kit provides an economical means of detecting proteins involved in NHEJ DNA repair. The kit includes enough antibodies to perform two western blot experiments with each primary antibody.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Background

DNA double-strand breaks (DSBs) are potentially hazardous lesions that can be induced by ionizing radiation (IR), radiomimetic chemicals, or DNA replication inhibitors. Cells recognize and repair DSBs via two distinct but partly overlapping signaling pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). DNA repair via the HR pathway is restricted to S and G2 phases of the cell cycle, while NHEJ can occur during any phase. NHEJ machinery is also utilized in V(D)J recombination, a process that generates diversity in immunoglobulin and T cell receptor genes. Defects in both pathways have been associated with human disease, including cancer (1).
DNA repair through the NHEJ pathway involves a core group of proteins that includes the Ku heterodimer (Ku70/Ku80), DNA-PKcs, DNA ligase IV, XRCC4, and XLF. XLF interacts with XRCC4 and promotes the ligation of DNA strands by DNA ligase IV and the ligase cofactor XRCC4. The ATP-dependent ligation of free DNA ends is the final step in the NHEJ repair pathway (2). DNA ligase IV and the endonuclease artemis suppress homologous recombination and promote NHEJ (3).

  1. Hartlerode, A.J. and Scully, R. (2009) Biochem J 423, 157-68.
  2. Tsai, C.J. et al. (2007) Proc Natl Acad Sci U S A 104, 7851-6.
  3. Kurosawa, A. et al. (2013) PLoS One 8, e72253.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

    Limited Uses

    Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

    Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

    Revision 1
    #76696

    Non-Homologous End Joining (NHEJ) DNA Repair Antibody Sampler Kit

    Non-Homologous End Joining (NHEJ) DNA Repair Antibody Sampler Kit: Image 1 Expand Image
    Western blot analysis of extracts from various cell lines using Artemis (D7O8V) Rabbit mAb.
    Non-Homologous End Joining (NHEJ) DNA Repair Antibody Sampler Kit: Image 2 Expand Image
    Western blot analysis of extracts from various cell lines using DNA Ligase IV (D5N5N) Rabbit mAb.
    Non-Homologous End Joining (NHEJ) DNA Repair Antibody Sampler Kit: Image 3 Expand Image
    Western blot analysis of cell extracts from HeLa, A549 and COS cells using Ku80 (C48E7) Rabbit mAb.
    Non-Homologous End Joining (NHEJ) DNA Repair Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts of various cell lines using XLF Antibody.
    Non-Homologous End Joining (NHEJ) DNA Repair Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from M059K cells, which express normal levels of DNA-PKcs, and M059J cells, which lack DNA-PKcs, using DNA-PKcs (E6U3A) Rabbit mAb (upper) or α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).
    Non-Homologous End Joining (NHEJ) DNA Repair Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from various cell lines using Ku70 (D10A7) Rabbit mAb.
    Non-Homologous End Joining (NHEJ) DNA Repair Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts from M059K (DNA-PKcs positive) and M059J (DNA-PKcs negative) cells, untreated (-), or treated with neocarzinostatin (0.5 µg/ml, 1 hr; +) to induce DNA double strand breaks, using Phospho-DNA-PKcs (Ser2056) (E9J4G) Rabbit mAb (upper), total DNA-PKcs (E6U3A) Rabbit mAb #38168 (middle), or α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).
    Non-Homologous End Joining (NHEJ) DNA Repair Antibody Sampler Kit: Image 8 Expand Image
    After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
    Non-Homologous End Joining (NHEJ) DNA Repair Antibody Sampler Kit: Image 9 Expand Image
    Immunoprecipitation of Artemis from HT-29 cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Artemis (D7O8V) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot was performed using Artemis (D7O8V) Rabbit mAb.
    Non-Homologous End Joining (NHEJ) DNA Repair Antibody Sampler Kit: Image 10 Expand Image
    Immunohistochemical analysis of paraffin-embedded human glioblastoma using Ku80 (C48E7) Rabbit mAb.
    Non-Homologous End Joining (NHEJ) DNA Repair Antibody Sampler Kit: Image 11 Expand Image
    Immunohistochemical analysis of paraffin-embedded human ductal breast carcinoma using DNA-PKcs (E6U3A) Rabbit mAb.
    Non-Homologous End Joining (NHEJ) DNA Repair Antibody Sampler Kit: Image 12 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Ku80 (C48E7) Rabbit mAb.
    Non-Homologous End Joining (NHEJ) DNA Repair Antibody Sampler Kit: Image 13 Expand Image
    Immunohistochemical analysis of paraffin-embedded normal human colon using DNA-PKcs (E6U3A) Rabbit mAb.
    Non-Homologous End Joining (NHEJ) DNA Repair Antibody Sampler Kit: Image 14 Expand Image
    Immunohistochemical analysis of paraffin-embedded human melanoma using Ku80 (C48E7) Rabbit mAb.
    Non-Homologous End Joining (NHEJ) DNA Repair Antibody Sampler Kit: Image 15 Expand Image
    Immunohistochemical analysis of paraffin-embedded human renal cell carcinoma using DNA-PKcs (E6U3A) Rabbit mAb.
    Non-Homologous End Joining (NHEJ) DNA Repair Antibody Sampler Kit: Image 16 Expand Image
    Immunohistochemical analysis of paraffin-embedded human GIST using Ku80 (C48E7) Rabbit mAb.
    Non-Homologous End Joining (NHEJ) DNA Repair Antibody Sampler Kit: Image 17 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung squamous cell carcinoma using DNA-PKcs (E6U3A) Rabbit mAb.
    Non-Homologous End Joining (NHEJ) DNA Repair Antibody Sampler Kit: Image 18 Expand Image
    Immunohistochemical analysis of paraffin-embedded human endometriod carcinoma using DNA-PKcs (E6U3A) Rabbit mAb.
    Non-Homologous End Joining (NHEJ) DNA Repair Antibody Sampler Kit: Image 19 Expand Image
    Confocal immunofluorescent analysis of HeLa cells using Ku80 (C48E7) Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red).
    Non-Homologous End Joining (NHEJ) DNA Repair Antibody Sampler Kit: Image 20 Expand Image
    Immunohistochemical analysis of paraffin-embedded M059K cell pellet (left, positive) or M059J cell pellet (right, negative) using DNA-PKcs (E6U3A) Rabbit mAb.
    Non-Homologous End Joining (NHEJ) DNA Repair Antibody Sampler Kit: Image 21 Expand Image
    Immunoprecipitation of Ku80 from HeLa cells. Lane 1 is 10% input, lane 2 is precipitated with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Ku80 (C48E7) Rabbit mAb, #2180. Western blot was performed using Ku80 (C48E7) Rabbit mAb, #2180.
    Non-Homologous End Joining (NHEJ) DNA Repair Antibody Sampler Kit: Image 22 Expand Image
    Confocal immunofluorescent analysis of M059K cells (left, positive) and M059J cells (right, negative) using DNA-PKcs (E6U3A) Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red).
    Non-Homologous End Joining (NHEJ) DNA Repair Antibody Sampler Kit: Image 23 Expand Image
    Flow cytometric analysis of M059J cells (blue) and M059K cells (green) using DNA-PKcs (E6U3A) Rabbit mAb (solid lines) or a concentration matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.