Name: Anti-p95/NBS1 antibody [Y112]
Brand: RabMAb
SKU: ab32074
Rating: 4 (5 reviews)

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Western blot - Anti-p95/NBS1 antibody [Y112] (ab32074)

Key features and details

Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
Rabbit monoclonal [Y112] to p95/NBS1
Suitable for: WB, IHC-P
Knockout validated
Reacts with: Human

Carrier Free

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, IHC-Pmore details
Unsuitable for: Flow Cyt or ICC/IF
Species reactivity

Reacts with: Human
Predicted to work with: Mouse, Rat
Immunogen

Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
Positive control
- WB: HeLa, Jurkat and Wild-type A431 cell lysates. IHC-P: Human testis and skin carcinoma tissues.
General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
For more information see here.
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.

Form

Liquid
Storage instructions

Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
Storage buffer

pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Concentration information loading...
Purity

Protein A purified
Clonality

Monoclonal
Clone number

Y112
Isotype

IgG
Research areas

Alternative Versions
- Anti-p95/NBS1 antibody [Y112] - BSA and Azide free (ab220217)
ChIP Related Products
- ChIP Kit (ab500)
Compatible Secondaries
Conjugation kits
- PE / R-Phycoerythrin Conjugation Kit - Lightning-Link® (ab102918)
- APC Conjugation Kit - Lightning-Link® (ab201807)
Isotype control
- Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
KO cell lines
- Human NBN (p95/NBS1) knockout HeLa cell line (ab261834)
- Human NBN (p95/NBS1) knockout A-431 cell line (ab269506)
KO cell lysates
- Human NBN (p95/NBS1) knockout HeLa cell lysate (ab257111)
- Human NBN (p95/NBS1) knockout A-431 cell lysate (ab269668)
Positive Controls
Recombinant Protein
- Recombinant Human p95/NBS1 protein (ab158956)

The Abpromise guarantee

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	(3)	1/1000 - 1/10000. Predicted molecular weight: 85 kDa. For unpurified use at 1/1000 - 1/2000.
IHC-P	(1)	1/200. Perform heat mediated antigen retrieval before commencing with IHC staining protocol. See IHC antigen retrieval protocols. For unpurified use at 1/50.

Notes
WB 1/1000 - 1/10000. Predicted molecular weight: 85 kDa. For unpurified use at 1/1000 - 1/2000.
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.

Application notes

Is unsuitable for Flow Cyt or ICC/IF.

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.
Target information above from: UniProt accession O60934 The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010) .

Information by UniProt
Database links
- Entrez Gene: 4683 Human
- Entrez Gene: 27354 Mouse
- Entrez Gene: 85482 Rat
- Omim: 602667 Human
- SwissProt: O60934 Human
- SwissProt: Q9R207 Mouse
- SwissProt: Q9JIL9 Rat
- Unigene: 492208 Human
- Unigene: 20866 Mouse
- Unigene: 25214 Rat
see all
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

Western blot - Anti-p95/NBS1 antibody [Y112] (ab32074)

All lanes : Anti-p95/NBS1 antibody [Y112] (ab32074) at 1/1000 dilution

Lane 1 : Wild-type A431 cell lysate
Lane 2 : NBN knockout A431 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 85 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?

Lanes 1 - 2: Merged signal (red and green). Green - ab32074 observed at 90 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab32074 was shown to react with p95/NBS1 in wild-type A431 cells in Western blot with loss of signal observed in NBN knockout cell line ab269506 (NBN knockout cell lysate ab269668). Wild-type A431 and NBN knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5 % milk in TBS-T (0.1 % Tween^®) before incubation with ab32074 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.
Western blot - Anti-p95/NBS1 antibody [Y112] (ab32074)

All lanes : Anti-p95/NBS1 antibody [Y112] (ab32074) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : NBN knockout HeLa cell lysate
Lane 3 : Wild-type HeLa nuclear cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

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

Lanes 1-3: Merged signal (red and green). Green - ab32074 observed at 95 kDa.

ab32074 Anti-p95/NBS1 antibody [Y112] was shown to specifically react with p95/NBS1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab261834 (knockout cell lysate ab257111) was used. Wild-type and p95/NBS1 knockout samples were subjected to SDS-PAGE. ab32074, Anti-GAPDH antibody [6C5] - Cytoplasmic Loading Control (ab8245) and Anti-Histone H3 (ab176842) - Nuclear Loading Control were incubated overnight at 4°C at 1 in 1000 dilution, 1 in 20000 dilution and 1 in 1000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773), Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) at 1 in 10000 dilution for 1 hour at room temperature before imaging.
Western blot - Anti-p95/NBS1 antibody [Y112] (ab32074)

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) - Anti-p95/NBS1 antibody [Y112] (ab32074)

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.
Western blot - Anti-p95/NBS1 antibody [Y112] (ab32074)

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) - Anti-p95/NBS1 antibody [Y112] (ab32074)

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.
Anti-p95/NBS1 antibody [Y112] (ab32074)

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

SDS download

Country/Region

Language
Datasheet download

Download

Publishing research using ab32074? Please let us know so that we can cite the reference in this datasheet.

ab32074 has been referenced in 25 publications.

Zhang B et al. NBS1 is required for SPO11-linked DNA double-strand break repair in male meiosis. Cell Death Differ 27:2176-2190 (2020). PubMed: 31965061
Leimbacher PA et al. MDC1 Interacts with TOPBP1 to Maintain Chromosomal Stability during Mitosis. Mol Cell 74:571-583.e8 (2019). PubMed: 30898438
Carvajal-Maldonado D et al. Perturbing cohesin dynamics drives MRE11 nuclease-dependent replication fork slowing. Nucleic Acids Res 47:1294-1310 (2019). PubMed: 29917110
Afzal S et al. Rat Glioma Cell-Based Functional Characterization of Anti-Stress and Protein Deaggregation Activities in the Marine Carotenoids, Astaxanthin and Fucoxanthin. Mar Drugs 17:N/A (2019). PubMed: 30909572
Hwang SY et al. CTCF cooperates with CtIP to drive homologous recombination repair of double-strand breaks. Nucleic Acids Res 47:9160-9179 (2019). PubMed: 31340001

View all Publications for this product

Customer reviews and Q&As

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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

The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted Apr 24 2013

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

The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted May 30 2014

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

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

The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

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

The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Abcam user community

Verified customer

Submitted May 19 2010

Question

Inquiry: i wanted to know the concentration of tha antibody Anti-p95 NBS1 ChIP grade (ab32074) lot: GR 4228-7 thanks in advance

Abcam community

Verified customer

Asked on Jun 22 2012

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.

Abcam Scientific Support

Answered on Jun 22 2012

Question

what is the concentration of Anti-p95 NBS1 antibody [Y112] - ChIP Grade (ab32074) lot GR4228-5?

Abcam community

Verified customer

Asked on May 02 2012

Answer

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

Abcam Scientific Support

Answered on May 02 2012

Question

Batch variability in ChIP. Would like to replace vials from alot that did not work well in ChIPwith vials from adifferent lot.

Abcam community

Verified customer

Asked on Mar 26 2012

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.

Abcam Scientific Support

Answered on Mar 26 2012

Question

Dear Madam / Sir,

A user needs a information about positive control tissue p95 NBS1 antibody [Y112] (ab32074). It is stated that Human skin carcinoma as positive control tissue on the datasheet. But It isn't clear which types of skin cancer. Could you please clear that?

Thank you.

Best regards,

Abcam community

Verified customer

Asked on Mar 08 2012

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.

Abcam Scientific Support

Answered on Mar 08 2012

Question

What is the concentration of this antibody, lot #GR50908-2?

Abcam community

Verified customer

Asked on Mar 06 2012

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.

Abcam Scientific Support

Answered on Mar 06 2012

Question

Lot GR4228-7 showed enrichment for negative control gene with mouse lymphocyte samples. Is lot GR50908-2 better for ChIP?

Abcam community

Verified customer

Asked on Mar 01 2012

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.

Abcam Scientific Support

Answered on Mar 01 2012

1-10 of 12 Abreviews or Q&A

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Key features and details

Related conjugates and formulations

Overview

Product name

Description

Host species

Tested applications

Species reactivity

Immunogen

Positive control

General notes

Properties

Form

Storage instructions

Storage buffer

Purity

Clonality

Clone number

Isotype

Research areas

Associated products

Alternative Versions

ChIP Related Products

Compatible Secondaries

Conjugation kits

Isotype control

KO cell lines

KO cell lysates

Positive Controls

Recombinant Protein

Applications

The Abpromise guarantee

Target

Function

Tissue specificity

Involvement in disease

Sequence similarities

Domain

Post-translationalmodifications

Cellular localization

Database links

Alternative names

Images

Protocols

Datasheets and documents

SDS download

Datasheet download

References (25)

Customer reviews and Q&As

Western blot abreview for Anti-p95 NBS1 antibody [Y112]

Immunocytochemistry/ Immunofluorescence abreview for Anti-p95 NBS1 antibody [Y112]

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-p95/NBS1 antibody [Y112]

Western blot abreview for Anti-p95 NBS1 antibody [Y112]

Post-translational
modifications