Overview

  • Product name
    Anti-p95/NBS1 antibody [Y112]
    See all p95/NBS1 primary antibodies
  • Description
    Rabbit monoclonal [Y112] to p95/NBS1
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, IP, IHC-P, ChIP/Chipmore details
    Unsuitable for: Flow Cyt or ICC/IF
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human p95/NBS1 aa 650-750 (C terminal). The exact sequence is proprietary.
    Database link: O60934

  • Positive control
    • WB: HeLa and Jurkat cell lysates. IHC-P: Human testis and skin carcinoma tissues. ICC/IF: HeLa cells. Flow Cyt: HeLa cells.
  • General notes

     

     

     This product was previously labelled as p95 NBS1

     

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab32074 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB 1/1000 - 1/10000. Predicted molecular weight: 85 kDa.

For unpurified use at 1/1000 - 1/2000.

IP 1/60.
IHC-P 1/200. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

For unpurified use at 1/50.

ChIP/Chip Use at an assay dependent concentration. PubMed: 21255732
  • Application notes
    Is unsuitable for Flow Cyt or ICC/IF.
  • Target

    • Function
      Component of the MRE11-RAD50-NBN (MRN complex) which plays a critical role in the cellular response to DNA damage and the maintenance of chromosome integrity. The complex is involved in double-strand break (DSB) repair, DNA recombination, maintenance of telomere integrity, cell cycle checkpoint control and meiosis. The complex possesses single-strand endonuclease activity and double-strand-specific 3'-5' exonuclease activity, which are provided by MRE11A. RAD50 may be required to bind DNA ends and hold them in close proximity. NBN modulate the DNA damage signal sensing by recruiting PI3/PI4-kinase family members ATM, ATR, and probably DNA-PKcs to the DNA damage sites and activating their functions. It can also recruit MRE11 and RAD50 to the proximity of DSBs by an interaction with the histone H2AX. NBN also functions in telomere length maintenance by generating the 3' overhang which serves as a primer for telomerase dependent telomere elongation. NBN is a major player in the control of intra-S-phase checkpoint and there is some evidence that NBN is involved in G1 and G2 checkpoints. The roles of NBS1/MRN encompass DNA damage sensor, signal transducer, and effector, which enable cells to maintain DNA integrity and genomic stability. Forms a complex with RBBP8 to link DNA double-strand break sensing to resection. Enhances AKT1 phosphorylation possibly by association with the mTORC2 complex.
    • Tissue specificity
      Ubiquitous. Expressed at high levels in testis.
    • Involvement in disease
      Nijmegen breakage syndrome
      Breast cancer
      Aplastic anemia
      Defects in NBN might play a role in the pathogenesis of childhood acute lymphoblastic leukemia (ALL).
    • Sequence similarities
      Contains 1 BRCT domain.
      Contains 1 FHA domain.
    • Domain
      The FHA and BRCT domains are likely to have a crucial role for both binding to histone H2AFX and for relocalization of MRE11/RAD50 complex to the vicinity of DNA damage.
      The C-terminal domain contains a MRE11-binding site, and this interaction is required for the nuclear localization of the MRN complex.
      The EEXXXDDL motif at the C-terminus is required for the interaction with ATM and its recruitment to sites of DNA damage and promote the phosphorylation of ATM substrates, leading to the events of DNA damage response.
    • Post-translational
      modifications
      Phosphorylated by ATM in response of ionizing radiation, and such phosphorylation is responsible intra-S phase checkpoint control and telomere maintenance.
    • Cellular localization
      Nucleus. Nucleus, PML body. Chromosome, telomere. Localizes to discrete nuclear foci after treatment with genotoxic agents.
    • Information by UniProt
    • Database links
    • Alternative names
      • AT V1 antibody
      • AT V2 antibody
      • ATV antibody
      • Cell cycle regulatory protein p95 antibody
      • FLJ10155 antibody
      • MGC87362 antibody
      • Nbn antibody
      • NBN_HUMAN antibody
      • NBS 1 antibody
      • NBS antibody
      • NBS1 antibody
      • Nibrin antibody
      • Nijmegen breakage syndrome 1 (nibrin) antibody
      • Nijmegen breakage syndrome antibody
      • Nijmegen breakage syndrome protein 1 antibody
      • p95 antibody
      • p95 protein of the MRE11/RAD50 complex antibody
      see all

    Images

    • All lanes : Anti-p95/NBS1 antibody [Y112] (ab32074) at 1/1200 dilution (unpurified)

      Lane 1 : HeLa cell lysate
      Lane 2 : Jurkat cell lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

      Predicted band size: 85 kDa
      Observed band size: 95 kDa
      why is the actual band size different from the predicted?



      Blocking buffer and concentration: 5% NFDM/TBST.

      Diluting buffer and concentration: 5% NFDM /TBST.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human testis tissue labelling p95/NBS1 with purified ab32074 at 1/200. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Counterstained with hematoxylin.

    • All lanes : Anti-p95/NBS1 antibody [Y112] (ab32074) at 1/10000 dilution (purified)

      Lane 1 : HeLa cell lysate
      Lane 2 : Jurkat cell lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

      Predicted band size: 85 kDa
      Observed band size: 95 kDa why is the actual band size different from the predicted?



      Blocking buffer and concentration: 5% NFDM/TBST.

      Diluting buffer and concentration: 5% NFDM /TBST.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human testis tissue labelling p95/NBS1 with unpurified ab32074 at 1/20. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Counterstained with hematoxylin.

    References

    This product has been referenced in:
    • Carvajal-Maldonado D  et al. Perturbing cohesin dynamics drives MRE11 nuclease-dependent replication fork slowing. Nucleic Acids Res 47:1294-1310 (2019). Read more (PubMed: 29917110) »
    • Huang D  et al. Isoorientin triggers apoptosis of hepatoblastoma by inducing DNA double-strand breaks and suppressing homologous recombination repair. Biomed Pharmacother 101:719-728 (2018). WB ; Human . Read more (PubMed: 29524880) »
    See all 20 Publications for this product

    Customer reviews and Q&As

    1-10 of 12 Abreviews or Q&A

    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HeLa)
    Gel Running Conditions
    Reduced Denaturing (4-12% Bis-Tris)
    Loading amount
    50 µg
    Treatment
    10 Gy IR
    Specification
    HeLa
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

    Abcam user community

    Verified customer

    Submitted Apr 24 2013

    This product is known to not work in this application or species.
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (HeLa)
    Permeabilization
    Yes - Triton X-100
    Specification
    HeLa
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5%
    Fixative
    Formaldehyde

    Abcam user community

    Verified customer

    Submitted May 30 2014

    Answer

    Thank you for your inquiry.

    I am happy to confirm that the concentration of lot GR4228-7 is 0.645 mg/mL.

    I also would like to comment on the seemingly low concentration:

    The IgG produced by rabbits have greater affinity and greater sensitivity than mouse IgG (about 100-1000 fold) and at the same time the total amount of IgG produced by the rabbit hybridomas is generally lower than in mouse hybridomas.

    Many 'veteran' antibody users are generally familiar with the IgG amount provided from mouse antibodies and can judge how many reactions they may get or need to use based on concentration. Unfortunately, this experience is not transferable to rabbit monoclonal antibodies because the concentration scales are generally very different.

    Since the specificity and sensitivity is much higher with rabbit monoclonal antibodies, less IgG is required per reaction.

    I would like to reassure you that while the amount of IgG may not be comparable to mouse monoclonal antibodies, customers are receiving a comparable number of reactions/tests/blots/stainings per tube.

    I hope this information is helpful. Please do not hesitate to contact me with any further questions in this matter.

    Read More

    Answer


    The concentration of ab32074 lot GR4228-5is : 0.645 mg/mL.

    Read More

    Answer

    Thank you for your call today and for keeping me updated about this situation.

    I am sending the three vials thatwe havein stock. These vials will be arriving on the free of charge order 1060035, and I expect them to arrive tomorrow.

    I've also issued credit note***for one vial of ab32074. The full purchase price will be refunded back to your credit card pending approval from our accounting department. If you have any questions about this credit note, please contact mailto:us.credits@abcam.com.

    I apologize again for the batch variability of this antibody. Please let me know if you have any questions or if there is anything else that we can do for you.

    Read More

    Answer

    Thank you for your enquiry.

    I can confirm that p95 NBS1 antibody [Y112] (ab32074) was tested onsquamous cell carcinoma of the skin. This would be a suitable positive control.

    I hope this information will be helpful to you. If you have any further questions, please do not hesitate to contact us.

    Read More

    Question
    Answer

    Thank you for contacting Abcam.



    The concentration for ab32074, lot #GR50908-2 is *****mg/mL.



    Please let me know if there is anything else I can help you with.

    Read More

    Answer

    Thanks for your call today and for letting us know about the trouble with ab32074 in ChIP.

    As we discussed, I am sending a free of charge vial of ab32074 from lot GR50908-2 on the order ***, which should arrive tomorrow.

    I've looked through our records, and we do not test this antibody in-house in ChIP. This application was added to our datasheet based on results in the following article-

    http://www.ncbi.nlm.nih.gov/pubmed/21255732?dopt=Abstract

    So unfortunately I don't have any data for each of these lots in ChIP, but I am hopeful that the new vial will work better for you. Please keep me updated about the results with this vial, and let me know if you have any questions or need anything else from me.

    Read More
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Human Tissue sections (Human carotid plaque tissue)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: Citrate buffer
    Permeabilization
    No
    Specification
    Human carotid plaque tissue
    Blocking step
    Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Fixative
    Formaldehyde

    Abcam user community

    Verified customer

    Submitted Nov 23 2011

    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (Jurkat cells)
    Gel Running Conditions
    Reduced Denaturing
    Loading amount
    100000 cells
    Specification
    Jurkat cells
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

    Abcam user community

    Verified customer

    Submitted May 19 2010

    1-10 of 12 Abreviews or Q&A

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