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Anti-53BP1 antibody [EPR2172(2)] ab175933 is a rabbit monoclonal antibody that is used in 53BP1 western blotting, IHC, immunofluorescence and flow cytometry. Suitable for human, mouse and rat samples.

- Recombinant format for unrivaled batch-batch consistency: no need for same-lot requests
- Specificity confirmed with TP53BP1 knockout cell line validation
- Specificity and sensitivity confirmed in IHC with multi-tissue microarray (TMA) validation
- Antibody clone EPR2172(2) is cited in over 60 publications


Images

Immunocytochemistry/ Immunofluorescence - Anti-53BP1 antibody [EPR2172(2)] (AB175933), expandable thumbnail
  • Western blot - Anti-53BP1 antibody [EPR2172(2)] (AB175933), expandable thumbnail
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-53BP1 antibody [EPR2172(2)] (AB175933), expandable thumbnail
  • Flow Cytometry (Intracellular) - Anti-53BP1 antibody [EPR2172(2)] (AB175933), expandable thumbnail
  • Western blot - Anti-53BP1 antibody [EPR2172(2)] (AB175933), expandable thumbnail

Publications

Key facts

Isotype

IgG

Host species

Rabbit

Storage buffer

Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA

Form

Liquid

Clonality

Monoclonal

Immunogen

  • The exact immunogen used to generate this antibody is proprietary information.

Reactivity data

Select an application
Product promiseTestedExpectedPredictedNot recommended
IHC-PWBICC/IFFlow Cyt (Intra)
Human
Tested
Tested
Tested
Tested
Mouse
Tested
Tested
Expected
Expected
Rat
Tested
Tested
Expected
Expected

Tested
Tested

Species

Mouse

Dilution info

1/60 - 1/250

Notes

Perform heat-mediated antigen retrieval before commencing with IHC staining protocol.

Species

Rat

Dilution info

1/60 - 1/250

Notes

Perform heat-mediated antigen retrieval before commencing with IHC staining protocol.

Species

Human

Dilution info

1/60 - 1/250

Notes

Perform heat-mediated antigen retrieval before commencing with IHC staining protocol.

Tested
Tested

Species

Mouse

Dilution info

1/1000 - 1/5000

Notes

-

Species

Rat

Dilution info

1/1000 - 1/5000

Notes

-

Species

Human

Dilution info

1/1000 - 1/5000

Notes

-

Tested
Tested

Species

Human

Dilution info

1/100 - 1/250

Notes

-

Expected
Expected

Species

Mouse, Rat

Dilution info

Use at an assay dependent concentration.

Notes

-

Tested
Tested

Species

Human

Dilution info

-

Notes

-

Expected
Expected

Species

Mouse, Rat

Dilution info

Use at an assay dependent concentration.

Notes

-

Associated Products

Select an associated product type

7 products for Alternative Product

Target data

Function

Double-strand break (DSB) repair protein involved in response to DNA damage, telomere dynamics and class-switch recombination (CSR) during antibody genesis (PubMed:12364621, PubMed:22553214, PubMed:23333306, PubMed:17190600, PubMed:21144835, PubMed:28241136). Plays a key role in the repair of double-strand DNA breaks (DSBs) in response to DNA damage by promoting non-homologous end joining (NHEJ)-mediated repair of DSBs and specifically counteracting the function of the homologous recombination (HR) repair protein BRCA1 (PubMed:22553214, PubMed:23727112, PubMed:23333306). In response to DSBs, phosphorylation by ATM promotes interaction with RIF1 and dissociation from NUDT16L1/TIRR, leading to recruitment to DSBs sites (PubMed:28241136). Recruited to DSBs sites by recognizing and binding histone H2A monoubiquitinated at 'Lys-15' (H2AK15Ub) and histone H4 dimethylated at 'Lys-20' (H4K20me2), two histone marks that are present at DSBs sites (PubMed:23760478, PubMed:28241136, PubMed:17190600). Required for immunoglobulin class-switch recombination (CSR) during antibody genesis, a process that involves the generation of DNA DSBs (PubMed:23345425). Participates in the repair and the orientation of the broken DNA ends during CSR (By similarity). In contrast, it is not required for classic NHEJ and V(D)J recombination (By similarity). Promotes NHEJ of dysfunctional telomeres via interaction with PAXIP1 (PubMed:23727112).

Alternative names

TP53-binding protein 1, 53BP1, p53-binding protein 1, p53BP1, TP53BP1

Recommended products

Anti-53BP1 antibody [EPR2172(2)] ab175933 is a rabbit monoclonal antibody that is used in 53BP1 western blotting, IHC, immunofluorescence and flow cytometry. Suitable for human, mouse and rat samples.

- Recombinant format for unrivaled batch-batch consistency: no need for same-lot requests
- Specificity confirmed with TP53BP1 knockout cell line validation
- Specificity and sensitivity confirmed in IHC with multi-tissue microarray (TMA) validation
- Antibody clone EPR2172(2) is cited in over 60 publications

Key facts

Isotype

IgG

Form

Liquid

Clonality

Monoclonal

Immunogen
  • The exact immunogen used to generate this antibody is proprietary information.
Clone number

EPR2172(2)

Purification technique

Affinity purification Protein A

Concentration
Loading...

Storage

Shipped at conditions

Blue Ice

Appropriate short-term storage duration

1-2 weeks

Appropriate short-term storage conditions

+4°C

Appropriate long-term storage conditions

-20°C

Aliquoting information

Upon delivery aliquot

Storage information

Avoid freeze / thaw cycle

Notes

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

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 batch production

For more information, read more on recombinant antibodies.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

53BP1 also known as p53-binding protein 1 is a protein that plays an important role in DNA damage response. The 53BP1 protein has a molecular weight of approximately 213 kDa. It is a nuclear protein and it mainly expresses in cells during the DNA damage repair processes. Researchers commonly utilize immunofluorescence and anti-53BP1 antibodies to detect its presence especially for studying DNA repair mechanisms. 53BP1 proves important for maintenance of genomic stability.

Biological function summary

As a DNA damage response mediator 53BP1 functions to recognize and bind to sites of double-strand breaks in DNA promoting non-homologous end joining (NHEJ). This protein is a component of a larger protein complex that recruits factors necessary for DNA repair. Its interaction with DNA lesions also involves chromatin remodeling aiming to facilitate successful repair of the DNA. 53BP1 works in concert with other proteins such as H2AX and MDC1.

Pathways

Researchers observe 53BP1 in processes such as the DNA damage checkpoint and repair pathways. This protein significantly contributes to the pathways involved in maintaining cell cycle checkpoints. It interacts with proteins like ATM and BRCA1 coordinating the cellular response to DNA damage and ensuring that genomic integrity checks occur properly before cell cycle progression continues. These interactions place 53BP1 at a pivotal position in decision-making between repair pathways.

Associated diseases and disorders

Researchers often focus on 53BP1's involvement in cancer development and therapy resistance due to its role in DNA repair. Alterations or deficiencies in 53BP1 levels can contribute to increased susceptibility to tumor development affecting processes like breast cancer and ovarian cancer through its relationship with BRCA1. The improper function of 53BP1 in DNA repair pathways may lead to an accumulation of genetic mutations driving tumorigenesis and impacting the efficacy of certain cancer treatments.

Product promise

We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.

In the unlikely event of one of our products not working as expected, you are covered by our product promise.

Full details and terms and conditions can be found here:
Terms & Conditions.

23 product images

  • Immunocytochemistry/ Immunofluorescence - Anti-53BP1 antibody [EPR2172(2)] (ab175933), expandable thumbnail

    Immunocytochemistry/ Immunofluorescence - Anti-53BP1 antibody [EPR2172(2)] (ab175933)

    Immunocytochemistry/Immunofluorescence analysis of HepG2 cells labelling 53BP1 with purified ab175933 at 1/200. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, a mouse anti-tubulin (1/1000) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

    Control 1: primary antibody (1/200) and secondary antibody, Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000).

    Control 2: Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (1/1000) and secondary antibody, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000).

  • Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933), expandable thumbnail

    Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933)

    Lane 1: Wild-type HAP1 cell lysate (20 μg)
    Lane 2: 53BP1 knockout HAP1 cell lysate (20 μg)
    Lane 3: HeLa cell lysate (40 μg)
    Lane 4: HepG2 cell lysate (40 μg)

    Lanes 1 - 4: Merged signal (red and green). Green - ab175933 observed at 350 kDa. Red - loading control, ab18058, observed at 124 kDa.
    ab175933 was shown to specifically react with 53BP1 when 53BP1 knockout samples were used. Wild-type and 53BP1 knockout samples were subjected to SDS-PAGE. ab175933 and ab18058 (loading control to Vinculin) were diluted 1/1000 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

    All lanes: Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933)

    Predicted band size: 214 kDa

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-53BP1 antibody [EPR2172(2)] (ab175933), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-53BP1 antibody [EPR2172(2)] (ab175933)

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat liver tissue labelling 53BP1 with purified ab175933 at a dilution of 1/60. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. Goat Anti-Rabbit IgG H&L (HRP) ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

  • Flow Cytometry (Intracellular) - Anti-53BP1 antibody [EPR2172(2)] (ab175933), expandable thumbnail

    Flow Cytometry (Intracellular) - Anti-53BP1 antibody [EPR2172(2)] (ab175933)

    ab175933 staining 53BP1in the human cell lineHepG2 (human hepatocellular carcinoma) by intracellular flow cytometry. Cells were fixed with 4% paraformaldehyde and the sample was incubated with the primary antibody at a dilution of 1/30. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/2000 was used as the secondary antibody.

    Isoytype control: Rabbit monoclonal IgG (Black)

    Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue)

  • Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933), expandable thumbnail

    Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933)

    Lane 1: Wild-type HAP1 cell lysate (20 μg)
    Lane 2: 53BP1 knockout HAP1 cell lysate (20 μg)
    Lane 3: HeLa cell lysate (40 μg)
    Lane 4: HepG2 cell lysate (40 μg)

    Lanes 1 - 4: Merged signal (red and green).

    Green - Target observed at 350 kDa. Red - loading control, ab18058, observed at 124 kDa.

    This western blot image is a comparison between ab175933 and a competitor's top cited rabbit polyclonal antibody.

    All lanes: Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933)

    Predicted band size: 214 kDa

  • Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933), expandable thumbnail

    Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933)

    Blocking and dilution buffer: 5% NFDM /TBST.

    All lanes: Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933) at 1/1000 dilution

    Lane 1: Mouse heart tissue lysate at 10 µg

    Lane 2: Mouse brain tissue lysate at 10 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

    Predicted band size: 214 kDa

    Observed band size: 450 kDa

  • Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933), expandable thumbnail

    Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933)

    Blocking and dilution buffer: 5% NFDM /TBST.

    All lanes: Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933) at 20 µg

    All lanes: Rat heart tissue lysate at 20 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

    Predicted band size: 214 kDa

    Observed band size: 450 kDa

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-53BP1 antibody [EPR2172(2)] (ab175933), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-53BP1 antibody [EPR2172(2)] (ab175933)

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse liver tissue labelling 53BP1 with purified ab175933 at a dilution of 1/60. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. Goat Anti-Rabbit IgG H&L (HRP) ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

  • Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933), expandable thumbnail

    Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933)

    Blocking and dilution buffer: 5% NFDM /TBST.

    All lanes: Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933) at 1/5000 dilution

    Lane 1: Human fetal heart tissue lysate at 20 µg

    Lane 2: HeLa whole cell lysate at 20 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

    Predicted band size: 214 kDa

    Observed band size: 450 kDa

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-53BP1 antibody [EPR2172(2)] (ab175933), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-53BP1 antibody [EPR2172(2)] (ab175933)

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver carcinoma tissue labelling 53BP1 with purified ab175933 at a dilution of 1/60. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. Goat Anti-Rabbit IgG H&L (HRP) ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

  • Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933), expandable thumbnail

    Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933)

    All lanes: Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933) at 1/1000 dilution

    Lane 1: HepG2 cell lysate at 1/10 dilution

    Lane 2: Human fetal brain lysate at 1/10 dilution

    Lane 3: Human fetal heart lysate at 1/10 dilution

    Predicted band size: 214 kDa

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-53BP1 antibody [EPR2172(2)] (ab175933), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-53BP1 antibody [EPR2172(2)] (ab175933)

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human tonsil tissue labelling 53BP1 with unpurified ab175933 at a dilution of 1/100.

    Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-53BP1 antibody [EPR2172(2)] (ab175933), expandable thumbnail

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-53BP1 antibody [EPR2172(2)] (ab175933)

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon tissue labelling 53BP1 with unpurified ab175933 at a dilution of 1/100.

    Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

  • Immunocytochemistry/ Immunofluorescence - Anti-53BP1 antibody [EPR2172(2)] (ab175933), expandable thumbnail

    Immunocytochemistry/ Immunofluorescence - Anti-53BP1 antibody [EPR2172(2)] (ab175933)

    Immunocytochemistry/Immunofluorescence analysis of HepG2 cells labelling 53BP1 with unpurified ab175933 at a dilution of 1/100.

  • Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933), expandable thumbnail

    Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933)

    Blocking and diluting buffer and concentration: 5% NFDM/TBST.

    In Western blot, Anti-Vinculin antibody [EPR8185] (Anti-Vinculin antibody [EPR8185] ab129002) staining at 1/10000 dilution.

    In Western blot, Anti-53BP1 antibody [EPR2172(2)] (ab175933) staining at 350kDa dilution.

    All lanes: Western blot - Anti-53BP1 (phospho T670) antibody [EPR27060-30] (Anti-53BP1 (phospho T670) antibody [EPR27060-30] ab314866) at 1/1000 dilution

    Lane 1: Untreated wild-type HAP1(human chronic myelogenous leukemia near-haploid cell line) whole cell lysate (untreated membrane) at 20 µg

    Lane 2: Wild-type HAP1 treated with 100nM Calycin A for 30 minutes, whole cell lysate (untreated membrane) at 20 µg

    Lane 3: Untreated TP53BP1 knockout HAP1 whole cell lysate (untreated membrane) at 20 µg

    Lane 4: TP53BP1 knockout HAP1 treated with 100nM Calycin A for 30 minutes, whole cell lysate (untreated membrane) at 20 µg

    Lane 5: Untreated wild-type HAP1(human chronic myelogenous leukemia near-haploid cell line) whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

    Lane 6: Wild-type HAP1 treated with 100nM Calycin A for 30 minutes, whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

    Lane 7: Untreated TP53BP1 knockout HAP1 whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

    Lane 8: TP53BP1 knockout HAP1 treated with 100nM Calycin A for 30 minutes, whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

    Observed band size: 350 kDa, 124 kDa

    Exposure time: 180s

  • Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933), expandable thumbnail

    Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933)

    Blocking and diluting buffer and concentration: 5% NFDM/TBST

    The identity of the bands below 300 kDa is unknown.

    In Western blot, Anti-Vinculin antibody [EPR8185] (Anti-Vinculin antibody [EPR8185] ab129002) staining at 1/10000 dilution.

    In Western blot, Anti-53BP1 antibody [EPR2172(2)] (ab175933) staining at 1/1000 dilution.

    All lanes: Western blot - Anti-53BP1 (phospho S25) antibody [EPR27057-33] (Anti-53BP1 (phospho S25) antibody [EPR27057-33] ab315015) at 1/1000 dilution

    Lane 1: Untreated HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 µg

    Lane 2: HeLa treated with 20J/m2 UV, then recovery for 1 hour, whole cell lysate at 20 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

    Observed band size: 350 kDa, 124 kDa

    Exposure time: 10s

  • Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933), expandable thumbnail

    Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933)

    Blocking and diluting buffer and concentration: 5% NFDM/TBST.

    The identity of the bands below 300 kDa is unknown.

    In Western blot, Anti-Vinculin antibody [EPR8185] (Anti-Vinculin antibody [EPR8185] ab129002) staining at 1/10000 dilution.

    In Western blot, Anti-53BP1 antibody [EPR2172(2)] (ab175933) staining at 1/1000 dilution.

    All lanes: Western blot - Anti-53BP1 (phospho S25) antibody [EPR27057-33] (Anti-53BP1 (phospho S25) antibody [EPR27057-33] ab315015) at 1/1000 dilution

    Lane 1: Untreated wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell line) whole cell lysate (untreated membrane) at 20 µg

    Lane 2: Wild-type HAP1 treated with 100nM Calycin A for 30 minutes, whole cell lysate (untreated membrane) at 20 µg

    Lane 3: Untreated TP53BP1 knockout HAP1 whole cell lysate (untreated membrane) at 20 µg

    Lane 4: TP53BP1 knockout HAP1 treated with 100nM Calycin A for 30 minutes, whole cell lysate (untreated membrane) at 20 µg

    Lane 5: Untreated wild-type HAP1 whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

    Lane 6: Wild-type HAP1 treated with 100nM Calycin A for 30 minutes, whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

    Lane 7: Untreated TP53BP1 knockout HAP1 whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

    Lane 8: TP53BP1 knockout HAP1 treated with 100nM Calycin A for 30 minutes, whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

    Observed band size: 350 kDa, 124 kDa

    Exposure time: 3s

  • Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933), expandable thumbnail

    Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933)

    Blocking and diluting buffer and concentration: 5% NFDM/TBST.

    The identity of the bands below 300 kDa is unknown.

    Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

    In Western blot, Anti-Vinculin antibody [EPR8185] (Anti-Vinculin antibody [EPR8185] ab129002) staining at 1/10000 dilution.

    In Western blot, Anti-53BP1 antibody [EPR2172(2)] (ab175933) staining at 1/1000 dilution.

    All lanes: Western blot - Anti-53BP1 (phospho S25) antibody [EPR27057-33] (Anti-53BP1 (phospho S25) antibody [EPR27057-33] ab315015) at 1/1000 dilution

    Lane 1: Untreated U-2 OS (human bone osteosarcoma epithelial cell) whole cell lysate at 20 µg

    Lane 2: U-2 OS treated with 10um Camptothecin for 1 hour, whole cell lysate at 20 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

    Observed band size: 350 kDa, 124 kDa

    Exposure time: 3s

  • Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933), expandable thumbnail

    Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933)

    The identity of the band at 130 kDa is unknown.

    The bands beneath the target band (350 kDa) are likely to be degraded target fragments which are with total protein control ab175933.

    In Western blot, Anti-53BP1 (phospho T543) antibody [EPR27059-168] ab316316 was shown to bind specifically to 53BP1 (phospho T543). A band was observed at 350 kDa in Calyculin A treated wild-type HAP1 cell lysates whereas loss of signal was observed in the 53BP1 knockout cell line.

    In Western blot, Anti-Vinculin antibody [EPR8185] (Anti-Vinculin antibody [EPR8185] ab129002) staining at 1/10000 dilution.

    In Western blot, Anti-53BP1 antibody [EPR2172 (2)] (ab175933) staining at 1/1000 dilution.

    All lanes: Western blot - Anti-53BP1 (phospho T543) antibody [EPR27059-168] (Anti-53BP1 (phospho T543) antibody [EPR27059-168] ab316316) at 1/1000 dilution

    Lane 1: Untreated wild-type HAP1(human chronic myelogenous leukemia near-haploid cell line) whole cell lysate (untreated membrane) at 20 µg

    Lane 2: Wild-type HAP1 treated with 100nM Calycin A for 30 minutes, whole cell lysate (untreated membrane) at 20 µg

    Lane 3: Untreated TP53BP1 knockout HAP1 whole cell lysate (untreated membrane) at 20 µg

    Lane 4: TP53BP1 knockout HAP1 treated with 100nM Calycin A for 30 minutes, whole cell lysate (untreated membrane) at 20 µg

    Lane 5: Untreated wild-type HAP1(human chronic myelogenous leukemia near-haploid cell line) whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

    Lane 6: Wild-type HAP1 treated with 100nM Calycin A for 30 minutes, whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

    Lane 7: Untreated TP53BP1 knockout HAP1 whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

    Lane 8: TP53BP1 knockout HAP1 treated with 100nM Calycin A for 30 minutes, whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

    Observed band size: 350 kDa, 124 kDa

    Exposure time: 48s

  • Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933), expandable thumbnail

    Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933)

    The identity of the band at 130 kDa is unknown.

    The bands beneath the target band (350 kDa) are likely to be degraded target fragments which are with total protein control ab175933 .

    In Western blot, Anti-Vinculin antibody [EPR8185] (Anti-Vinculin antibody [EPR8185] ab129002) staining at 1/10000 dilution.

    In Western blot, Anti-53BP1 antibody [EPR2172 (2)] (ab175933) staining at 1/1000 dilution.

    All lanes: Western blot - Anti-53BP1 (phospho T543) antibody [EPR27059-168] (Anti-53BP1 (phospho T543) antibody [EPR27059-168] ab316316) at 1/1000 dilution

    Lane 1: Untreated U-2 OS (human bone osteosarcoma epithelial cell) whole cell lysate (untreated membrane) at 20 µg

    Lane 2: U-2 OS treated with 10um Camptothecin for 1 hour, whole cell lysate (untreated membrane) at 20 µg

    Lane 3: Untreated U-2 OS (human bone osteosarcoma epithelial cell) whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

    Lane 4: U-2 OS treated with 10um Camptothecin for 1 hour, whole cell lysate (alkaline phosphatase treated membrane) at 20 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

    Observed band size: 350 kDa, 124 kDa

    Exposure time: 48s

  • Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933), expandable thumbnail

    Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933)

    The identity of the band at 130 kDa is unknown.

    The bands beneath the target band (350 kDa) are likely to be degraded target fragments .

    In Western blot, Anti-Vinculin antibody [EPR8185] (Anti-Vinculin antibody [EPR8185] ab129002) staining at 1/10000 dilution.

    In Western blot, Anti-53BP1 antibody [EPR2172 (2)] (ab175933) staining at 1/1000 dilution.

    All lanes: Western blot - Anti-53BP1 (phospho T543) antibody [EPR27059-168] (Anti-53BP1 (phospho T543) antibody [EPR27059-168] ab316316) at 1/1000 dilution

    Lane 1: Untreated HeLa (human epithelial cell line from cervical adenocarcinoma) whole cell lysate at 20 µg

    Lane 2: HeLa treated with /ml Calyculin A for 15 minutes, whole cell lysate at 20 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

    Observed band size: 350 kDa, 124 kDa

    Exposure time: 180s

  • Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933), expandable thumbnail

    Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933)

    Blocking and diluting buffer and concentration: 5% NFDM/TBST.

    In Western blot, Anti-Vinculin antibody [EPR8185] (Anti-Vinculin antibody [EPR8185] ab129002) staining at 1/10000 dilution.

    In Western blot, Anti-53BP1 antibody [EPR2172(2)] (ab175933) staining at 350kDa dilution.

    All lanes: Western blot - Anti-53BP1 (phospho T670) antibody [EPR27060-30] (Anti-53BP1 (phospho T670) antibody [EPR27060-30] ab314866) at 1/1000 dilution

    Lane 1: Untreated HeLa (human cervix adenocarcinoma epithelial cell) at 20 µg

    Lane 2: HeLa treated with 20J/m2 UV, then recovery for 1 hour, whole cell lysate at 20 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

    Developed using the ECL technique.

    Observed band size: 350 kDa, 124 kDa

    Exposure time: 114s

  • Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933), expandable thumbnail

    Western blot - Anti-53BP1 antibody [EPR2172(2)] (ab175933)

    Blocking and diluting buffer and concentration: 5% NFDM/TBST.

    In Western blot, Anti-Vinculin antibody [EPR8185] (Anti-Vinculin antibody [EPR8185] ab129002) staining at 1/10000 dilution.

    In Western blot, Anti-53BP1 antibody [EPR2172(2)] (ab175933) staining at 350kDa dilution.

    All lanes: Western blot - Anti-53BP1 (phospho T670) antibody [EPR27060-30] (Anti-53BP1 (phospho T670) antibody [EPR27060-30] ab314866) at 1/1000 dilution

    Lane 1: HeLa (human epithelial cell line from cervical adenocarcinoma) whole cell lysate at 20 µg

    Lane 2: HeLa treated with 100ng/ml Calyculin A for 10 minutes, whole cell lysate at 20 µg

    Lane 3: A431 (human epidermoid carcinoma epithelial cell) whole cell lysate at 20 µg

    Lane 4: A431 treated with 100ng/ml Calyculin A for 15 minutes, whole cell lysate at 20 µg

    Secondary

    All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

    Observed band size: 350 kDa, 124 kDa

    Exposure time: 180s

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