TP53BP1 Antibody
Description
Definition and Biological Role of TP53BP1
TP53BP1 is a 214 kDa nuclear phosphoprotein encoded by the TP53BP1 gene (NCBI Gene ID: 7158) that facilitates DNA double-strand break (DSB) repair by regulating repair pathway choice between non-homologous end joining (NHEJ) and homologous recombination (HR) . It acts as a scaffold protein, recruiting downstream repair factors like Shieldin, RIF1, and PTIP to DSB sites . TP53BP1 also modulates immune responses in cancer by influencing tumor mutational burden and T-cell infiltration .
DNA Damage Response Studies
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TP53BP1 facilitates ATM-dependent phosphorylation of KAP-1, promoting chromatin relaxation for homologous recombination .
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Depletion of TP53BP1 disrupts G2/M checkpoint arrest and BRCA1 foci formation, critical for DSB repair .
Cancer Immunology
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Low TP53BP1 expression correlates with increased tumor mutational burden (TMB) and CD8+ T-cell infiltration in high-grade serous ovarian cancer (HGSOC) and pancreatic ductal adenocarcinoma (PDAC) .
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TP53BP1 loss sensitizes BRCA1-deficient tumors to immune checkpoint blockade (ICB) by activating the cGAS-STING pathway .
Mechanistic Insights
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TP53BP1 prevents DNA end resection by recruiting DYNLL1 and Shieldin, favoring NHEJ over HR .
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Its Tudor domain mediates binding to methylated histones (e.g., H4K20me2), anchoring it to damaged chromatin .
Clinical and Therapeutic Implications
Protocol Considerations
Product Specs
Target Background
- Inhibition of 53BP1 is an effective method for increasing the efficiency of HDR-based precise genome editing. PMID: 29176614
- This research elucidates the mechanism by which TIRR recognizes 53BP1 Tudor and functions as a cellular inhibitor of histone methyl-lysine readers. PMID: 29844495
- The data indicate the molecular mechanism underlying Tudor interacting repair regulator (TIRR)-mediated suppression of tumor protein p53 binding protein 1 (53BP1)-dependent DNA damage repair. PMID: 30002377
- GFI1 facilitates efficient DNA repair by regulating PRMT1 dependent methylation of MRE11 and 53BP1. PMID: 29651020
- The results suggest that TP53BP1 and MFN1 frameshift mutations and their intratumoral heterogeneity (ITH) could contribute to cancer development by inhibiting the TSG activities. PMID: 30082159
- The results highlight the interplay of RNF169 with 53BP1 in fine-tuning the choice of DSB repair pathways. PMID: 30104380
- Despite the requirement of all three nucleoporins for accurate NHEJ, only Nup153 is needed for proper nuclear import of 53BP1 and SENP1-dependent sumoylation of 53BP1. Data support the role of Nup153 as an important regulator of 53BP1 activity and efficient NHEJ. PMID: 28576968
- Results indicate that the integrity of the nuclear localization signal is important for 53BP1 nuclear localization. PMID: 29603287
- As shown in a xenograft model of glioblastoma, phosphorylation of 53BP1 by GSK3beta was indispensable for DNA double-strand break repair. PMID: 29328365
- Results suggest that there is a direct interaction between 53BP1 and MCMs, which is essential for 53BP1 chromatin fraction and foci formation in hepatoma HepG2 cells. PMID: 29990989
- Results indicate that 53BP1 is a biomarker of response to anti-PARP therapy in the laboratory, and our DNA damage response gene signature may be used to identify patients who are most likely to respond to PARP inhibition. PMID: 28958991
- These results reveal two distinct fork restart pathways, which are antagonistically controlled by 53BP1 and BRCA1 in a double-strand DNA break repair-independent manner. PMID: 29106372
- Gamma-H2AX, phosphorylated KAP-1 and 53BP1 play an important role in the repair of heterochromatic radon-induced DNA double-strand breaks. PMID: 27922110
- Data show that the expression of tumor protein p53 binding protein 1 (53BP1) varies at different stages of the cell cycle, with high-level expression observed in mitosis. PMID: 28930533
- Results further highlight the antagonistic relationship between 53BP1 and BRCA1, and place Nup153 and Nup50 in a molecular pathway that regulates 53BP1 function by counteracting BRCA1-mediated events. PMID: 28751496
- PAXIP1 and 53BP1 protein levels followed gene expression results, i.e., are intrinsically correlated, and also reduced in more advanced breast cancer tumors. PMID: 28475402
- Data indicate that p53-binding protein 1 (53BP1) is required to prevent excessive chromosome missegregation and probably genome hyper-instability, and also for optimal growth in cancer cells. PMID: 29445165
- Study demonstrates a consistent resistance profile to PARPi and a unique cross-resistance profile to non-PARPi drugs in different PARPi-resistant U251 glioblastoma cells and reveals 53BP1 loss and SAMHD1 overexpression as the primary mechanisms responsible for their resistance to PARPi and Ara-C, respectively. PMID: 29274141
- The number of gammaH2AX foci did not significantly change following cardiac MR (median foci per cell pre-MR = 0.11, post-MR = 0.11, p = .90), but the number of 53BP1 foci significantly increased following MR PMID: 29309426
- Premature maturation of post-replicative chromatin restores Histone h4 lysine 20 methylation and rescues 53BP1 accumulation on replicated chromatin. PMID: 28564601
- UVA-induced progerinlamin A complex formation was largely responsible for suppressing 53BP1-mediated NHEJ DSB repair activity. The present study is the first to demonstrate that UVA-induced progerin upregulation adversely affects 53BP1-mediated NHEJ DSB repair in human keratinocytes via progerinlamin A complex formation. PMID: 28498430
- 53BP1/RIF1 has a role in limiting BRCA1/CtIP-mediated end resection to control double strand break repair pathway choice PMID: 27494840
- It observed a distinct accumulation of 53BP1 protein to UV-induced DNA lesions: in R273C mutants, 53BP1 appeared transiently at DNA lesions, during 10-30 min after irradiation; the mutation R282W was responsible for accumulation of 53BP1 immediately after UVA-damage; and in L194F mutants, the first appearance of 53BP1 protein at the lesions occurred during 60-70 min. PMID: 28397142
- A reciprocal regulation between 53BP1 and APC/C that is required for response to mitotic stress. PMID: 28228263
- BRCA1 promotes PP4C-dependent 53BP1 dephosphorylation and RIF1 release, directing repair toward homologous recombination. PMID: 28076794
- Co-localization of gammaH2AX and 53BP1 indicates promotion of (in)effective nonhomologous end-joining repair mechanisms at sites of DSB. Moreover, gammaH2AX/53BP1 foci distribution presumably reveals a non-random spatial organization of the genome in MDS and AML. PMID: 28359030
- Results provide evidence that 53BP1 is involved in breast cancer cells resistance for PARP inhibitor; its depletion causes resistance in ATM-deficient tumor cells. PMID: 27613518
- Ubiquitin ligases RNF168, RNF169, and RAD18 specifically bind histone H2A Lys13/15-ubiquitylated nucleosomes. 53BP1 chromatin recruitment may be activated by RNF168 and blocked by RNF169 and RAD18. PMID: 28506460
- Ras-induced senescent cells are hindered in their ability to recruit BRCA1 and 53BP1 to DNA damage sites. Whereas BRCA1 is downregulated at transcripts levels, 53BP1 loss is caused by activation of cathepsin L-mediated degradation of 53BP1 protein. We discovered a marked downregulation of vitamin D receptor (VDR) during OIS, and a role for the vitamin D/VDR axis regulating the levels of these DNA repair PMID: 27041576
- TIP60 complex regulates bivalent chromatin recognition/modification by 53BP1 through direct H4K20me binding and H2AK15 acetylation. PMID: 27153538
- Findings identify TIRR as a new factor that influences double-strand break repair using a unique mechanism of masking the histone methyl-lysine binding function of 53BP1 PMID: 28241136
- Deficiency of 53BP1 inhibits the radiosensitivity of colorectal cancer. PMID: 27499037
- The interplay between 53BP1/NHEJ and BRCA1/HR is highly relevant for tumor treatment, as the 53BP1 status would be highly important for the treatment response of BRCA1-associated tumors. PMID: 26615718
- Exhaustion of 53BP1 by increasing the load of double strand breaks suppresses RAD51 accumulation in repair foci during S and G2. PMID: 27348077
- TIRR is a novel 53BP1-interacting protein that participates in the DNA damage response PMID: 28213517
- These data suggest that multiple pathways collectively fine-tune the cellular levels of 53BP1 protein to ensure proper DSB repair and cell survival. PMID: 28255090
- This shows that 53BP1 protects both close and distant DSEs from degradation and that the association of unprotection with distance between DSEs favors ECS capture. Reciprocally, silencing CtIP lessens ECS capture both in control and 53BP1-depleted cells. We propose that close ends are immediately/rapidly tethered and ligated, whereas distant ends first require synapsis of the distant DSEs prior to ligation PMID: 27798638
- Increased 53BP1 expression (i.e., "unstable" expression) in nuclear foci of oncocytic follicular adenoma (FA) of the thyroid correlates with a higher incidence of DNA copy numbers compared with conventional FA. PMID: 26935218
- High 53BP1 mRNA is associated with head and neck cancer. PMID: 27465548
- During live-cell imaging, 53BP1-GFP focus formation was observed within 10 minutes after UVC irradiation. Most 53BP1 foci resolved by 100 minutes. To block UVC-induced double-strand break repair in cancer cells, poly(ADP-ribose) polymerase (PARP) was targeted with ABT-888 (veliparib). PARP inhibition markedly enhanced UVC-irradiation-induced persistence of 53BP1-foci PMID: 27466483
- Combined effect of dynamic recruitment of RNF4 to KAP1 regulates the relative occupancy of 53BP1 and BRCA1 at double-strand break sites to direct DNA repair in a cell cycle-dependent manner. PMID: 26766492
- The formation of 53BP1, gammaH2AX foci and their co-localization induced by gamma-rays (2, 5, 10, 50, 200 cGy) in human lymphocytes, was analyzed. PMID: 26243567
- 5-Hydroxymethylcytosine (5hmC) accumulates at DNA damage foci and colocalizes with major DNA damage response proteins 53BP1 and gH2AX, revealing 5hmC as an epigenetic marker of DNA damage. PMID: 26854228
- We show that XIST and 53BP1 can be used to identify BRCA1-like breast cancer patients who have higher event rates and poor outcome after HD chemotherapy. PMID: 26637364
- The kinetics of the accumulation of selected DNA repair-related proteins is protein specific at locally induced DNA lesions, and the formation of gH2AX- and NBS1-positive foci, but not 53BP1-positive NBs, is cell cycle dependent in HeLa cells PMID: 26482424
- The interaction of 53BP1 with gammaH2AX is required for sustaining the 53BP1-dependent focal concentration of activated ATM that facilitates repair of DNA double-strand breaks in heterochromatin in G1. PMID: 26628370
- Cryo-EM structure of a dimerized human 53BP1 fragment bound to a H4K20me2-containing and H2AK15ub-containing nucleosome core particle at 4.5 A resolution PMID: 27462807
- The study shows higher expression of gamma-H2AX and 53BP1 foci in rectal cancer patients compared with healthy individuals. Yet the data in vitro were not predictive in regard to the radiotherapy outcome. PMID: 26541290
- For both, gamma-H2AX and 53BP1, the cellular focus number as well as the percentage of positive cells did not differ between patients with clinically isolated syndrome/early relapsing-remitting multiple sclerosis and healthy controls. PMID: 26820970
- Impaired TIP60-mediated H4K16 acetylation accounts for the aberrant chromatin accumulation of 53BP1 and RAP80 in Fanconi anemia pathway-deficient cells. PMID: 26446986
Q&A
What is TP53BP1 and why is it important in research?
TP53BP1 (tumor protein p53 binding protein 1), also known as 53BP1, is a critical DNA damage response protein involved in checkpoint signaling during mitosis. It enhances TP53-mediated transcriptional activation and plays a key role in the response to DNA damage by promoting non-homologous end joining (NHEJ)-mediated repair of double-strand breaks (DSBs) .
TP53BP1 is particularly important in research because:
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It serves as a marker for DNA damage response activation
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It counteracts homologous recombination repair protein BRCA1, creating a balance between repair pathways
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Its loss correlates with response to immune checkpoint blockade therapy and prognosis in certain cancers
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It forms discrete nuclear foci at DNA damage sites, making it an excellent visual marker for DSBs
What are the most common applications for TP53BP1 antibodies?
TP53BP1 antibodies are used in multiple research applications, each with specific recommended dilutions:
For optimal results, it is recommended to titrate antibodies in each specific experimental system. Most antibodies detect TP53BP1 at approximately 450 kDa in Western blot applications, though the calculated molecular weight is 214 kDa .
How do I optimize TP53BP1 antibody staining for immunofluorescence experiments?
For optimal TP53BP1 immunofluorescence staining:
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Fixation: Use 4% paraformaldehyde for 10-15 minutes at room temperature
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Permeabilization: 0.2-0.5% Triton X-100 for 5-10 minutes (critical for nuclear antigen access)
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Blocking: 5% BSA or 5-10% normal serum from the species of the secondary antibody
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Primary antibody: Dilute according to manufacturer's recommendation (typically 1:125-1:1000)
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Incubation time: Overnight at 4°C generally yields the best signal-to-noise ratio
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Antigen retrieval: For tissue sections, try TE buffer pH 9.0 (alternatively, citrate buffer pH 6.0)
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Counterstain: DAPI for nuclear visualization, which helps localize TP53BP1 foci
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Controls: Include a no-primary antibody control and ideally a TP53BP1 knockout sample
When visualizing DNA damage-induced foci, allow sufficient time after damage induction (typically visible from 5 minutes to several hours post-irradiation) .
How do I troubleshoot non-specific banding or poor signal in TP53BP1 Western blots?
Non-specific banding is a common issue with TP53BP1 antibodies due to the protein's large size and potential degradation products. To address this:
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Verify specificity: Use knockout or knockdown controls to confirm specific bands. Multiple studies have demonstrated specificity using TP53BP1 knockout HeLa cell lines
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Address high background issues:
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Improve signal detection:
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Ensure adequate protein loading (typically 20-50 μg total protein)
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Use lower percentage gels (6-8%) for better resolution of high molecular weight proteins
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Extend transfer time for large proteins (overnight at lower voltage)
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Consider using PVDF membranes instead of nitrocellulose for higher protein binding capacity
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Verify molecular weight:
How do TP53BP1 antibodies perform across different sample types and species?
TP53BP1 antibody performance varies across species and sample types:
For tissue samples, antigen retrieval methods significantly impact staining quality. TE buffer pH 9.0 is generally recommended, though citrate buffer pH 6.0 can be used as an alternative . For cell lines, HeLa cells consistently show strong reactivity across multiple antibodies and are often used as positive controls .
When working with non-human samples, validation experiments are strongly recommended as antibody performance can vary significantly between manufacturers.
How can I quantify TP53BP1 foci in immunofluorescence experiments?
Quantitative analysis of TP53BP1 foci requires:
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Image acquisition optimization:
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Use confocal microscopy for better resolution of individual foci
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Standardize exposure settings across all experimental conditions
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Capture z-stacks to ensure all nuclear foci are detected
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Analysis approaches:
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Manual counting: Suitable for smaller experiments, but subject to observer bias
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Semi-automated: ImageJ/FIJI with plugins like FindFoci or Spot Detection
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Fully automated: CellProfiler pipelines or commercial software (e.g., MetaMorph, Imaris)
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Parameters to measure:
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Number of foci per nucleus
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Size/intensity of individual foci
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Nuclear area occupied by foci
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Co-localization with other DNA damage markers (e.g., γ-H2AX)
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Analysis guidelines:
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Count at least 100-200 cells per condition for statistical significance
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Establish clear criteria for what constitutes a focus (minimum size/intensity)
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Report both average foci per cell and distribution of foci across the population
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Research has shown that untreated cells typically display 0-2 TP53BP1 foci per nucleus, while DNA-damaged cells can show 5-50 foci depending on damage severity .
How do TP53BP1 detection methods contribute to understanding DNA repair pathway choice?
TP53BP1 serves as a key regulator in the decision between homologous recombination (HR) and non-homologous end joining (NHEJ) repair pathways. Advanced research applications include:
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Pathway competition analysis: TP53BP1 counteracts BRCA1-mediated end resection, directing repair toward NHEJ rather than HR . By simultaneously detecting TP53BP1 and BRCA1 localization, researchers can analyze pathway competition at individual break sites.
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Cell cycle-dependent repair dynamics: Using dual staining with cell cycle markers (e.g., PCNA, Cyclin B1) and TP53BP1, researchers can determine how repair pathway choice varies across cell cycle phases.
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Chromatin context influence: TP53BP1 recruitment depends on histone marks, particularly H2AK15Ub and H4K20me2 . Multi-parameter imaging with these histone modifications provides insight into chromatin-dependent repair mechanisms.
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Live-cell dynamics: Using tagged TP53BP1 constructs (typically the minimal foci-forming region), researchers can track the temporal dynamics of repair focus assembly and disassembly in real-time.
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Post-translational modification analysis: Phospho-specific TP53BP1 antibodies enable detection of ATM-dependent phosphorylation events that regulate 53BP1 function and protein interactions .
Research has demonstrated that the loss of TP53BP1 in BRCA1-deficient cells can restore HR and confer resistance to PARP inhibitors, highlighting the clinical relevance of these pathway interactions .
How can TP53BP1 antibodies be used to assess genomic instability in cancer research?
TP53BP1 antibodies serve as powerful tools for evaluating genomic instability in cancer contexts:
What are the latest methodological advances in applying TP53BP1 antibodies for challenging experimental systems?
Recent methodological innovations have expanded TP53BP1 antibody applications in difficult experimental contexts:
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Single-cell analysis: Integration of TP53BP1 immunofluorescence with single-cell RNA-seq or mass cytometry enables correlation between DNA damage response and transcriptional or proteomic profiles at the individual cell level.
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High-throughput screening: Automated TP53BP1 foci detection in plate-based formats allows screening of compounds affecting DNA repair, with applications in drug discovery and synthetic lethality identification.
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Tissue microenvironment studies: Multiplex immunofluorescence combining TP53BP1 with immune cell markers and spatial transcriptomics reveals how DNA damage in tumor cells influences the surrounding immune microenvironment—particularly relevant given the connection between 53BP1 loss and immune checkpoint blockade response .
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3D culture systems and organoids: Optimized protocols for TP53BP1 detection in 3D cultures, typically involving:
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Extended fixation times (24-48 hours)
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Increased permeabilization (0.5-1% Triton X-100, 48-72 hours)
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Extended antibody incubation (48-72 hours)
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Clearing techniques for improved imaging depth
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Detection in limited or degraded samples: Modified immunoprecipitation approaches and proximity ligation assays enhance sensitivity when analyzing rare cell populations or FFPE tissues with potential antigen degradation.
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Combined genomic and protein analyses: Techniques like immuno-FISH allow simultaneous visualization of TP53BP1 protein localization and specific DNA sequences, revealing relationships between repair protein recruitment and genomic features.
These advanced methods expand TP53BP1 antibody utility beyond conventional applications, enabling more sophisticated analyses of DNA damage response mechanisms in complex biological systems.
