Revision 1

#99891Store at -20C

1 Kit

(9 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
ATM (D2E2) Rabbit mAb 2873 20 µl 350 kDa Rabbit IgG
Phospho-ATM (Ser1981) (D25E5) Rabbit mAb 13050 20 µl 350 kDa Rabbit IgG
Rad51 (D4B10) Rabbit mAb 8875 20 µl 37 kDa Rabbit IgG
BRCA1 (A8X9F) Rabbit mAb 14823 20 µl 220 kDa Rabbit IgG
BRCA2 (D9S6V) Rabbit mAb 10741 20 µl 380 kDa Rabbit IgG
Rad54 (D4W3Z) Rabbit mAb 15016 20 µl 84 kDa Rabbit IgG
p95/NBS1 (D6J5I) Rabbit mAb 14956 20 µl 95 kDa Rabbit IgG
Phospho-p95/NBS1 (Ser343) Antibody 3001 20 µl 95 kDa Rabbit 
CtIP (D76F7) Rabbit mAb 9201 20 µl 110 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 Homologous Recombination (HR) DNA Repair Antibody Sampler Kit provides an economical means of detecting proteins involved in HR 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). DSBs that arise during S or G2 phase are repaired via HR, using the replicated sister chromatid as a repair template (1). Activation of ATM by autophosphorylation on Ser1981 occurs in response to exposed DNA DSBs. ATM regulates various responses to DNA damage, including regulation of HR factors (2). Rad51 recombinase polymerizes and forms a filament along single-stranded DNA, mediating HR with the help of auxiliary proteins, including Rad54 and BRCA2 (3). BRCA2 has been shown to be required for localization of Rad51 to sites of DSBs, and cells lacking BRCA1 and BRCA2 cannot repair DSBs through HR (4). NBS1 is critical for HR, and requires CDK-dependent association with CtIP and subsequent phosphorylation by ATM at Ser278 and Ser343 (5-6).

  1. Hartlerode, A.J. and Scully, R. (2009) Biochem J 423, 157-68.
  2. Lee, J.H. and Paull, T.T. (2007) Oncogene 26, 7741-8.
  3. Sung, P. et al. (2003) J Biol Chem 278, 42729-32.
  4. Tutt, A. and Ashworth, A. (2002) Trends Mol Med 8, 571-6.
  5. Wang, H. et al. (2013) PLoS Genet 9, e1003277.
  6. Wen, J. et al. (2013) Oncogene 32, 4448-56.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

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    Revision 1
    #99891

    Homologous Recombination (HR) DNA Repair Antibody Sampler Kit

    Homologous Recombination (HR) DNA Repair Antibody Sampler Kit: Image 1 Expand Image

    Simple Western™ analysis of lysates (0.1 mg/mL) from 293 cells treated with UV (100 mJ/cm2; 2 Hour Recovery) using Phospho-ATM (Ser1981) (D25E5) Rabbit mAb #13050. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 66 - 440 kDa separation module.

    Homologous Recombination (HR) DNA Repair Antibody Sampler Kit: Image 2 Expand Image
    Western blot analysis of extracts from various cell lines using BRCA2 (D9S6V) Rabbit mAb (upper) or α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).
    Homologous Recombination (HR) DNA Repair Antibody Sampler Kit: Image 3 Expand Image
    Western blot analysis of extracts from HCT 116 cells, untreated (-) or treated with neocarzinostatin (NCS 10 μM, 1 hr; +), using Phospho-ATM (Ser1981) (D25E5) Rabbit mAb (upper) and ATM (D2E2) Rabbit mAb #2873 (lower).
    Homologous Recombination (HR) DNA Repair Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from HT-29, HeLa, and MCF7 cells using BRCA1 (A8X9F) Rabbit mAb.
    Homologous Recombination (HR) DNA Repair Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from various cell lines using p95/NBS1 (D6J5I) Rabbit mAb (upper) and α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).
    Homologous Recombination (HR) DNA Repair Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from various cell lines using Rad54 (D4W3Z) Rabbit mAb.
    Homologous Recombination (HR) DNA Repair Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts of HeLa, NCCIT and PYS2 cells using ATM (D2E2) Rabbit mAb.
    Homologous Recombination (HR) DNA Repair Antibody Sampler Kit: Image 8 Expand Image
    Western blot analysis of extracts from 293 cells, untreated or UV-treated (100 mJ/cm2, 1 hr), using Phospho-p95/NBS1 (Ser343) Antibody (upper) or p95/NBS1 (E8M3Q) XP® Rabbit mAb #81234 (lower).
    Homologous Recombination (HR) DNA Repair Antibody Sampler Kit: Image 9 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.
    Homologous Recombination (HR) DNA Repair Antibody Sampler Kit: Image 10 Expand Image
    Western blot analysis of extracts from various cell lines using Rad51 (D4B10) Rabbit mAb.
    Homologous Recombination (HR) DNA Repair Antibody Sampler Kit: Image 11 Expand Image
    Western blot analysis of extracts from various cell lines using CtIP (D76F7) Rabbit mAb.
    Homologous Recombination (HR) DNA Repair Antibody Sampler Kit: Image 12 Expand Image
    Western blot analysis of DLD1 cells, either wild type (WT) or BRCA2 knockout (-/-), using BRCA2 (D9S6V) Rabbit mAb (upper) or α-Actinin (D6F6) XP® Rabbit mAb (lower). DLD1 (WT) and DLD1 (-/-) cells were kindly provided by Dr. Judit Jimenez, Yale University, New Haven, CT.
    Homologous Recombination (HR) DNA Repair Antibody Sampler Kit: Image 13 Expand Image
    Immunoprecipitation of p95/NBS1 from HeLa cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or p95/NBS1 (D6J5I) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot was performed using p95/NBS1 (D6J5I) Rabbit mAb.
    Homologous Recombination (HR) DNA Repair Antibody Sampler Kit: Image 14 Expand Image
    Immunoprecipitation of Rad54 from 293T cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Rad54 (D4W3Z) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot was performed using Rad54 (D4W3Z) Rabbit mAb.
    Homologous Recombination (HR) DNA Repair Antibody Sampler Kit: Image 15 Expand Image
    Immunoprecipitation of Rad51 from HeLa cell extracts using Rad51 (D4B10) Rabbit mAb (lane 2). Western blot detection was performed using the same antibody. Lane 1 is 10% input.
    Homologous Recombination (HR) DNA Repair Antibody Sampler Kit: Image 16 Expand Image
    Confocal immunofluorescent analysis of SK-MEL-28 (left) and GM07166 (right) cells using p95/NBS1 (D6J5I) Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054.