Phospho-BRCA1 (Ser1423) Antibody
Description
Definition and Biological Context
Phospho-BRCA1 (Ser1423) Antibody is a polyclonal antibody that specifically recognizes BRCA1 phosphorylated at Ser1423, a modification induced by DNA damage agents such as ionizing radiation (IR), ultraviolet (UV) light, hydroxyurea (HU), and aphidicolin (APH) . BRCA1 is a tumor suppressor protein encoded by the BRCA1 gene, mutated in ~50% of familial breast cancers . Its phosphorylation at Ser1423 is mediated by the kinases ATR (ATM- and Rad3-related) and ATM (ataxia-telangiectasia mutated), with ATR playing a dominant role in response to UV, HU, and APH, while ATM partially contributes to IR-induced phosphorylation .
Role of Ser1423 Phosphorylation
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DNA Damage Response: Ser1423 phosphorylation occurs in response to replication stress (e.g., HU, APH) or DNA damage (e.g., IR, UV) .
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Kinase Dependence:
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Functional Impact: Phosphorylated BRCA1 forms nuclear foci at stalled replication forks, facilitating DNA repair and cell cycle checkpoint activation .
Antibody Validation Data
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Specificity: Recognizes endogenous BRCA1 only when phosphorylated at Ser1423, confirmed via λ phosphatase treatment and Ser→Ala mutagenesis .
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Detection Sensitivity:
Applications in Research
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Mechanistic Studies: Used to investigate BRCA1’s role in homologous recombination repair (HRR) and its interaction with PALB2/RAD51 .
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Clinical Biomarker Research: Detects BRCA1 activation status in tumor samples, potentially correlating with therapeutic responses .
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Kinase Signaling Pathways: Differentiates ATR- vs. ATM-dependent DNA damage responses .
Limitations and Considerations
Product Specs
Target Background
- We conclude that BRCA1 and BRCA2 could be used as clinicopathological biomarkers to evaluate the prognosis of digestive system cancers. PMID: 29126833
- RAP80-BRCA1 complex foci formation is regulated by USP13. BRCA1 plays a significant role in the DNA damage response. PMID: 28569838
- RANK/RANKL have been identified as critical regulators for BRCA1 mutation-driven breast cancer. Current prevention strategies for BRCA1 mutation carriers involve significant risks, highlighting the need for alternative, non-invasive approaches. PMID: 29241686
- Neither the patients nor the control subjects in this study exhibited germline hypermethylation of the BRCA1 and BRCA2 promoter regions analyzed. PMID: 29404838
- Male individuals carrying BRCA mutations show significantly lower QMAX compared to healthy men. BRCA1 patients generally present with larger prostate glands and higher PSA levels compared to BRCA2 patients. PMID: 28577930
- Our findings provide evidence that BRCA1 undergoes intronic premature polyadenylation (pPA) following large internal exons, and that N(6)-methyladenosine levels in this exon are reduced in pPA-activated breast cancer cells. PMID: 29362392
- The combination of immunohistochemical expression of BRCA1, ER, PR, and HER-2/neu, along with clinicopathological details, may be helpful in identifying individuals more likely to carry BRCA1 mutations, facilitating targeted genetic screening for these mutations. PMID: 29567881
- Methylation of BRCA1 was found to be significantly associated with tumor grade. PMID: 30049201
- The IRIS-driven metastatic mechanism stems from IRIS-dependent suppression of phosphatase and tensin homolog (PTEN) transcription, which disrupts the PI3K/AKT/GSK-3beta pathway, leading to prolyl hydroxylase-independent HIF-1alpha stabilization and activation in a normoxic environment. PMID: 30254159
- Both BRCA1 and BRCA2 mutations are linked to an increased risk of Prostate cancer (PC). Specifically, BRCA2 mutations are associated with a more aggressive PC phenotype, characterized by a higher likelihood of locally advanced and metastatic disease, and should be considered a prognostic marker linked to poorer survival. PMID: 29242595
- Among individuals carrying BRCA mutations (BRCA1 or BRCA2), the survival advantage associated with preventive mastectomy at age 25 is substantial. However, the anticipated benefit diminishes rapidly with increasing age at surgery. PMID: 28914396
- We observed a significant increase in the frequencies of TP53 (rs1042522 G/C), BRCA1 (rs71361504 -/GTT, rs3092986T/C) genotypes and alleles in polycystic ovary patients compared to controls. PMID: 29860059
- BRCA1 Interacting Protein COBRA1 facilitates adaptation to castrate-resistant growth conditions. PMID: 30036938
- This family study illustrates the intertwined cancer spectrum of hereditary breast and ovarian cancer (HBOC) and familial pancreatic cancer (FPC) in BRCA1 families, raising awareness of the importance of considering pancreatic (head) adenocarcinoma (PAC) as a potential phenotypic representation of the HBOC tumor spectrum. (Fig. 1a) and one pancreatic (head) adenocarcinoma (PAC) PMID: 28900739
- High BRCA1 promoter methylation is linked to tumor grade and lymph node metastasis in breast cancer. PMID: 29970689
- This study demonstrates a clear protective effect of early first pregnancy on breast cancer risk in both BRCA1 and BRCA2 mutation carriers. PMID: 29116468
- BRCA1 deficiency was recurrent in early-onset triple-negative breast cancer in Brazilian patients and associated with improved survival. PMID: 29116469
- Overall, 5152 oncogenetic tests were reviewed in this study, of which 4452 had no a priori known familial mutation. The majority of participants (68.6%) underwent genotyping due to a personal history of cancer; 20.6% were tested based on family history of cancer, and details for the remaining 10.7% were missing. A total of 256/4452 (5.8%) carriers were detected, including 141 BRCA1 and 115 BRCA2 mutation carriers. PMID: 29086229
- CLDN3 expression and negative EGFR expression are associated with BRCA1 mutations in triple-negative breast cancers. PMID: 30142017
- This study aimed to elucidate the clinicopathological features, including the level of p53 protein expression and BRCA mutations, of primary fallopian tube cancer (PFTC) in Japanese women. PMID: 29982601
- Our findings suggest that BRCA1/2 germline mutations in China exhibit distinct characteristics compared to those in Western populations. PMID: 29681614
- Analysis confirmed the association between BRCA1 promoter methylation and breast cancer in Asia. PMID: 29693332
- A novel electrochemical DNA (E-DNA) biosensing strategy was designed and utilized for the detection of the breast cancer susceptibility gene (BRCA-1). PMID: 29698810
- Data suggests that targeting the breast cancer 1, early onset protein (BRCA1)-ribonucleotide reductase regulatory subunit M2 (RRM2) axis may represent a promising approach for therapeutic intervention in glioblastoma (GBM). PMID: 27845331
- We demonstrate a strong association between Triple Negative Breast Cancer and mutations in BRCA1/2 genes, as well as a poor prognosis for these patients. Survival curve analysis revealed that the presence of AKT1, TP53, KDR, KIT, BRCA1, and BRCA2 mutations is correlated with a poorer prognosis. PMID: 29202330
- Germline Mutation in the BRCA1 3'UTR Variant is associated with Breast Cancer. PMID: 29582646
- Homozygous loss of function BRCA1 variant causes a Fanconi-anemia-like phenotype. PMID: 29133208
- In summary, Nestin was strongly associated with germline BRCA1 related breast cancer, a basal-like phenotype, reduced survival, and stemness characteristics. PMID: 28439082
- Homozygous nonsense mutations in the tumor suppressor gene BRCA1 are associated with breast and ovarian cancer. PMID: 29712865
- Low BRCA1 expression is associated with radioresistance of glioma. PMID: 29286157
- BRCA1 germ line mutation is associated with unilateral triple-negative breast cancer. PMID: 29514593
- BRCA1 germ line mutation is associated with ovarian cancer. PMID: 29506471
- High Promoter Methylation of BRCA1 gene is associated with Breast Cancer. PMID: 29480000
- Ewing sarcoma cells exhibit alterations in the regulation of damage-induced transcription, accumulation of R-loops, and increased replication stress. Homologous recombination is impaired in Ewing sarcoma due to an enriched interaction between BRCA1 and the elongating transcription machinery. Furthermore, a role is identified for EWSR1 in the transcriptional response to damage, suppressing R-loops and promoting homologous recombination. PMID: 29513652
- Data indicate that BRCA1/2 mutations are not uncommon among selected Jordanian females with breast cancer. PMID: 29409476
- Data show that male BRCA1/2 mutation carriers with breast and prostate cancer demonstrated a favorable 5-year survival. PMID: 29433453
- Our findings suggest that gBRCA1/2 mutation was not associated with survival in Chinese EOC patients, potentially attributed to more than 37% of the patients without gross residual disease. Survival benefit of gBRCA1/2 mutation was prominent in ovarian cancer patients with gross residual disease. PMID: 29975922
- BRCA1 SNP rs1799950 is associated with enhanced response rate to pegylated liposomal doxorubicin in high-grade serous ovarian carcinomas. PMID: 29298688
- The results of Ion PGM with OTG-snpcaller, a pipeline based on Torrent mapping alignment program and Genome Analysis Toolkit, from 75 clinical samples and 14 reference DNA samples were compared with Sanger sequencing for BRCA1/BRCA2. PMID: 28392550
- Reduced BRCA1 expression was associated with ER and PR negative status resulting in Breast Carcinoma. PMID: 29286222
- In this study, we employed comprehensive multigene panels that included 35 known or suspected cancer susceptibility genes to examine BRCA1/2 mutation-negative Korean patients who exhibited clinical features suggestive of hereditary breast cancer. PMID: 29338689
- Pre-menopausal BRCA1/2 mutation carriers aged 30 to 47 years opted for screening, risk-reducing salpingo-oophorectomy (RRSO), or bilateral salpingectomy/delayed oophorectomy (BS/DO). For those undergoing BS/DO, delayed oophorectomy was recommended at age 40 years for BRCA1 and age 45 years for BRCA2 patients. PMID: 29735278
- Based on a cumulative risk of 0.55% to age 35 for BRCA1 mutation carriers and 0.56% to age 45 for BRCA2 mutation carriers, we recommend bilateral salpingo-oophorectomy before age 40, but by age 35 for women with a BRCA1 mutation and by age 45 for those with a BRCA2 mutation to maximize prevention and minimize adverse effects. PMID: 29793803
- Our findings demonstrate that homologous recombination deficiency (HRD) mutation signatures may provide clinically relevant information independent of BRCA1/2 mutation status, and we hope this work will guide the development of clinical trials. PMID: 29246904
- Overall, 65/648 (10%) study participants were BRCA1/2 mutation carriers. PMID: 30061222
- BRCA1*R1699Q confers an intermediate risk for breast cancer and ovarian cancer. PMID: 28490613
- Patient-derived xenografts effectively capture the molecular and phenotypic heterogeneity of triple-negative breast cancer. This study shows that PARP inhibition can exhibit activity beyond germline BRCA1/2 altered tumors, leading to regression in a variety of molecular subtypes. These models provide an opportunity for the discovery of rational combinations with targeted therapies and predictive biomarkers. PMID: 29093017
- BRCA methylation is infrequent in breast and ovarian carcinomas of BRCA germline mutation carriers, although the prevalence of BRCA promoter methylation might be underestimated. This finding has significant implications for clinical practice, including referrals for genetic testing and BRCAness analysis to inform treatment decision-making. PMID: 29891109
- Carboplatin and talazoparib demonstrated efficacy in DNA damage mutation carriers, but hematologic toxicity was more pronounced in gBRCA (gBRCA1/2) carriers. Carboplatin is most effectively combined with intermittent talazoparib dosing, differentiated based on germline and somatic DNA damage mutation carriers. PMID: 28790114
- Putative BRCA1/2 reversion mutations can be detected through cfDNA sequencing analysis in patients with ovarian and breast cancer. Our findings warrant further investigation of cfDNA sequencing to identify putative BRCA1/2 reversion mutations and guide the selection of patients for PARP inhibition therapy. PMID: 28765325
Q&A
What is the specificity of Phospho-BRCA1 (Ser1423) Antibody?
The Phospho-BRCA1 (Ser1423) Antibody specifically recognizes the breast cancer type 1 susceptibility protein (BRCA1), also known as RING finger protein 53 (RNF53), only when phosphorylated at serine 1423. This antibody is designed to detect endogenous levels of BRCA1 protein exclusively in its phosphorylated state at this specific residue . The specificity can be confirmed by several methods, including treatment with lambda phosphatase (which removes phosphorylation and eliminates antibody recognition) or by using BRCA1 with Ser1423Ala mutation, which prevents recognition by the antibody . The antibody's specificity makes it valuable for studying BRCA1 phosphorylation events in response to various DNA damaging agents.
What applications is the Phospho-BRCA1 (Ser1423) Antibody suitable for?
The Phospho-BRCA1 (Ser1423) Antibody can be used in multiple experimental applications:
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Western Blotting (WB): Typically used at dilutions of 1:500-1:2,000
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Immunohistochemistry (IHC): Recommended dilutions of 1:100-1:300
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Immunofluorescence (IF): Typically used at dilutions of 1:50-1:200
The antibody successfully detects a 210-220 kDa protein in human samples that corresponds to phosphorylated BRCA1 . When using this antibody for these applications, researchers should optimize the dilution ratios based on their specific experimental conditions and sample types.
How should Phospho-BRCA1 (Ser1423) Antibody be stored and handled?
For optimal preservation of antibody activity and specificity:
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Long-term storage: Store undiluted at -20°C. Avoid frost-free freezers as temperature cycling can damage the antibody
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Avoid repeated freeze-thaw cycles which can lead to loss of antibody activity
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If precipitate forms, microcentrifugation before use is recommended
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The antibody is typically supplied in phosphate buffered saline (PBS) with 50% glycerol and 0.02% sodium azide, which helps maintain stability
Proper storage and handling of the antibody is crucial for maintaining its specificity and sensitivity in detecting phosphorylated BRCA1 at Ser1423.
What is the reactivity spectrum of Phospho-BRCA1 (Ser1423) Antibody?
The Phospho-BRCA1 (Ser1423) Antibody primarily reacts with human BRCA1 . Some commercially available versions also show reactivity with rat BRCA1 . When planning experiments using this antibody, researchers should verify species cross-reactivity, especially if working with model organisms other than human cell lines or tissues. The antibody recognizes the specific phosphorylation site in the context of the surrounding amino acid sequence (HGsQP, where "s" represents the phosphorylated serine) . This sequence conservation is important for cross-species reactivity.
How does DNA damage affect BRCA1 phosphorylation at Ser1423 and how can it be detected with this antibody?
BRCA1 phosphorylation at Ser1423 is significantly enhanced in response to various types of DNA damage. Research has shown that:
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Ionizing radiation (IR): Induces rapid phosphorylation of BRCA1 at Ser1423, with ATM playing a significant role in this process
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Ultraviolet (UV) light: Triggers Ser1423 phosphorylation primarily through ATR kinase activity
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Hydroxyurea (HU): Causes replication stress that leads to increased Ser1423 phosphorylation
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Aphidicolin (APH): Inhibits DNA polymerase and enhances BRCA1 phosphorylation at Ser1423
When designing experiments to detect damage-induced phosphorylation, researchers should consider:
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Time course analysis: Phosphorylation typically occurs rapidly after damage and may show different kinetics depending on the damaging agent
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Dose-response relationships: Titration of damaging agents (e.g., 10-50 Gy for IR) can provide insight into sensitivity thresholds
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Cell cycle considerations: Asynchronously cycling cells show different baseline levels of phosphorylation
The Phospho-BRCA1 (Ser1423) Antibody can effectively detect these changes in phosphorylation status via Western blotting, allowing researchers to quantify differences between treatment conditions through densitometric analysis .
What is the role of ATM and ATR kinases in BRCA1 Ser1423 phosphorylation and how can this be studied?
The phosphorylation of BRCA1 at Ser1423 is regulated by two key DNA damage response kinases: ATM (Ataxia Telangiectasia Mutated) and ATR (ATM and Rad3-related). Their distinct roles can be studied using the Phospho-BRCA1 (Ser1423) Antibody:
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ATM primarily mediates IR-induced phosphorylation of BRCA1 at Ser1423
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ATR is primarily responsible for UV and replication stress-induced phosphorylation
Experimental approaches to study these relationships include:
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Using ATM-deficient and ATM-reconstituted cell lines to compare phosphorylation levels
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Employing inducible expression systems for wild-type or kinase-dead ATR
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Combining specific kinase inhibitors with DNA damaging agents
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Studying ATR relocalization to nuclear foci that overlap with BRCA1 foci after DNA damage
The Phospho-BRCA1 (Ser1423) Antibody is essential for these experiments as it allows direct measurement of phosphorylation levels in response to experimental manipulations.
How can in vitro kinase assays be performed to study BRCA1 Ser1423 phosphorylation?
In vitro kinase assays provide mechanistic insights into direct phosphorylation of BRCA1 by specific kinases. For studying Ser1423 phosphorylation:
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Substrate preparation:
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Kinase source:
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Reaction conditions:
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Combine purified kinase with GST-BRCA1 substrate in appropriate buffer
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Include ATP (often radiolabeled for detection purposes)
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Incubate at 30°C for 15-30 minutes
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Detection methods:
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For radiolabeled assays: SDS-PAGE followed by autoradiography
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For non-radioactive assays: Western blotting with Phospho-BRCA1 (Ser1423) Antibody
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Compare phosphorylation by wild-type versus kinase-inactive mutants
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Research has shown that ATR specifically phosphorylates GST-BRCA1 fragments containing Ser1423 in vitro, and this phosphorylation is absent when using catalytically inactive ATR mutants . These assays help establish direct kinase-substrate relationships and validate the specificity of phosphorylation events observed in cells.
How can immunohistochemistry with Phospho-BRCA1 (Ser1423) Antibody be optimized for cancer tissue analysis?
Optimizing immunohistochemistry (IHC) protocols for Phospho-BRCA1 (Ser1423) Antibody in cancer tissues requires careful consideration of several factors:
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Tissue preparation and fixation:
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Use fresh frozen or properly fixed (typically 10% neutral buffered formalin) tissues
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Optimal fixation time (usually 24-48 hours) is crucial to preserve phospho-epitopes
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Paraffin-embedded tissues require appropriate antigen retrieval methods
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Antigen retrieval methods:
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Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)
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Optimization of retrieval time and temperature is essential for phospho-epitopes
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Enzymatic retrieval may be less suitable for phospho-epitopes
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Blocking and antibody incubation:
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Detection and controls:
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Use sensitive detection systems (HRP/DAB, polymer-based systems)
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Include positive controls (breast carcinoma tissues with known BRCA1 phosphorylation)
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Include negative controls (tissues treated with lambda phosphatase)
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Consider using BRCA1-deficient tissues (e.g., HCC1937 cells) as specificity controls
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Result interpretation:
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Analyze subcellular localization (typically nuclear for phosphorylated BRCA1)
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Quantify staining intensity and percentage of positive cells
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Compare with normal adjacent tissue when analyzing tumor samples
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Successful IHC with this antibody can reveal important insights into BRCA1 phosphorylation status in cancer tissues, potentially correlating with DNA damage response activity and treatment response.
