BRIP1 Antibody, Biotin conjugated

What is BRIP1 and why is it an important research target?

BRIP1 is a DNA-dependent helicase and 5'-3' DNA helicase required for maintaining chromosomal stability . It plays a crucial role in the Fanconi anemia pathway, acting after FANCD2 ubiquitination . BRIP1 functions in DNA repair processes, particularly in resolving DNA double-strand breaks through homologous recombination in association with BRCA1 . The protein is essential for unwinding G-quadruplex DNA structures that can form during lagging-strand DNA synthesis, thereby preventing genomic instability that could lead to tumorigenesis . Its interaction with BRCA1 and its location on chromosome 17q22 near the BRCA1 locus has made it a candidate tumor suppressor gene, with mutations being linked to increased breast cancer susceptibility .

What are the common applications for BRIP1 antibodies in research?

BRIP1 antibodies, including biotin-conjugated versions, are employed in multiple research techniques:

• Western Blotting (WB): Used to detect BRIP1 protein in cell lysates, with the protein appearing at approximately 140 kDa

• Immunoprecipitation (IP): For isolation and concentration of BRIP1 from complex biological samples

• Immunofluorescence (IF): For cellular localization studies of BRIP1, which typically shows nuclear localization

• Immunohistochemistry (IHC): For detection of BRIP1 in tissue sections, particularly in cancer tissues such as breast cancer samples

• ELISA: For quantitative measurement of BRIP1 in research samples

The biotin-conjugated format offers enhanced sensitivity through biotin-streptavidin detection systems, which amplify signals in various applications .

What cell lines have been validated for BRIP1 antibody applications?

Several human cell lines have been validated for use with BRIP1 antibodies:

These cell lines provide suitable positive controls for experimental design when working with BRIP1 antibodies . In breast cancer tissue samples, BRIP1 staining is characteristically localized to cell nuclei, consistent with its role in DNA repair processes .

How can the phosphorylation status of BRIP1 be monitored using biotin-conjugated antibodies?

The phosphorylation of BRIP1 at Serine 990 is crucial for its interaction with BRCA1 and subsequent function in DNA damage-induced checkpoint control during the G2/M phase of the cell cycle . To monitor this phosphorylation:

• Use antibodies that specifically recognize phosphorylated Ser990 in combination with biotin-conjugated antibodies through a dual staining approach

• Perform co-immunoprecipitation experiments using biotin-conjugated BRIP1 antibodies for protein capture, followed by phospho-specific detection methods

• Apply functional analysis tests comparing wild-type and phospho-mutant proteins in transfected cells

The cell cycle-regulated phosphorylation of BRIP1 can be detected by treating cells with DNA damaging agents and then examining phosphorylation levels using combined immunoprecipitation and western blotting techniques, where biotin-conjugated antibodies provide enhanced sensitivity and reduced background .

What experimental approaches can detect mutated BRIP1 in patient samples and assess functional implications?

Detecting BRIP1 mutations and assessing their functional impact requires multi-faceted approaches:

• Mutation Detection:

  • PCR amplification of the BRIP1 gene, with products ranging between 214-380 base pairs

  • Single Strand Conformation Polymorphism (SSCP) analysis for screening

  • Bidirectional sequencing to confirm mutations

• Functional Assessment:

  • Co-immunoprecipitation assays to evaluate BRIP1-BRCA1 interaction capacity

  • Protein stability assays in transfected cells with recombinant mutant proteins

  • Loss of heterozygosity analysis in tumor samples compared to normal tissue

One significant mutation (c.2992-2995delAAGA) identified in a woman with early-onset breast cancer caused a shift in the reading frame, disrupted the BRCA1-binding domain, and created a premature stop codon . Functional analysis demonstrated that this truncation interfered with both protein stability and BRCA1 interaction .

How can biotin-conjugated BRIP1 antibodies be utilized to investigate the relationship between BRIP1 and DNA repair mechanisms?

Biotin-conjugated BRIP1 antibodies offer advantages in investigating DNA repair mechanisms:

• DNA Damage Response Pathway Analysis:

  • ChIP (Chromatin Immunoprecipitation) assays using biotin-conjugated antibodies to identify DNA binding sites

  • Biotin-streptavidin pull-down systems for examining protein complexes formed at DNA damage sites

  • Co-localization studies with γ-H2AX (marker of DNA double-strand breaks)

• G-quadruplex Resolution Studies:

  • Immunoprecipitation of BRIP1 from cells treated with G-quadruplex stabilizing agents

  • Analysis of BRIP1 recruitment to replication forks using biotin-conjugated antibodies in chromatin fractionation experiments

• BRCA1-BRIP1 Complex Formation:

  • Sequential immunoprecipitation studies to isolate intact complexes

  • Investigation of cell cycle-dependent interactions

BRIP1 is particularly important in resolving secondary structures of guanine-rich DNA that form during lagging-strand DNA synthesis and is involved in repairing abasic sites at replication forks by promoting the degradation of DNA-protein cross-links .

What are the optimal conditions for Western blot detection of BRIP1 using biotin-conjugated antibodies?

For optimal Western blot detection of BRIP1 using biotin-conjugated antibodies:

A specific band for BRIP1 should be detected at approximately 140 kDa . If experiencing weak signals, consider:

• Increasing antibody concentration

• Extending incubation time to overnight at 4°C

• Using enhanced chemiluminescence substrates

• Implementing signal amplification methods that exploit the biotin-streptavidin interaction

How can biotin-conjugated BRIP1 antibodies be effectively used in immunohistochemistry of paraffin-embedded tissues?

For effective immunohistochemistry using biotin-conjugated BRIP1 antibodies on paraffin-embedded tissues:

• Preparation Protocol:

  • Subject tissue sections to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic

  • Apply antibody at 15 μg/mL concentration

  • Incubate overnight at 4°C for optimal binding

• Detection Strategy:

  • Utilize streptavidin-based detection systems (e.g., HRP-DAB)

  • Counterstain with hematoxylin for nuclear contrast

  • Look for specific nuclear staining, as BRIP1 predominantly localizes to nuclei

• Controls Implementation:

  • Include breast cancer tissue sections as positive controls

  • Use isotype-matched irrelevant antibodies as negative controls

  • Consider dual staining with BRCA1 antibodies to validate co-localization

When optimizing the protocol, remember that BRIP1 staining is predominantly nuclear due to its function in DNA repair mechanisms. The biotin conjugation allows for signal amplification through streptavidin systems, which is particularly valuable for detecting low-abundance proteins in tissue sections .

What are the main challenges and solutions when using biotin-conjugated BRIP1 antibodies in co-immunoprecipitation studies?

Challenges and solutions for co-immunoprecipitation with biotin-conjugated BRIP1 antibodies:

When investigating BRIP1-BRCA1 interactions, consider that phosphorylation of BRIP1 at Serine 990 is essential for this interaction . Therefore, maintaining phosphorylation status during experimental procedures is crucial for obtaining physiologically relevant results.

How can biotin-conjugated BRIP1 antibodies contribute to breast cancer research?

Biotin-conjugated BRIP1 antibodies offer valuable tools for breast cancer research:

• Genetic Predisposition Studies:

  • Detection of BRIP1 protein levels in samples from patients with BRIP1 mutations

  • Correlation of protein expression with mutation status and clinical outcomes

• Loss of Heterozygosity Analysis:

  • Comparison of BRIP1 protein levels between tumor and matched normal tissue

  • Investigation of whether wild-type or mutant BRIP1 alleles are retained in tumors

• Therapeutic Response Monitoring:

  • Evaluation of BRIP1 expression before and after DNA damage-inducing therapies

  • Assessment of DNA repair pathway activity through BRIP1 nuclear localization patterns

BRIP1 has been implicated as a breast cancer susceptibility gene, with a study identifying a novel germ-line mutation (c.2992-2995delAAGA) in a woman with early-onset breast cancer . The mutation disrupted the BRCA1-binding domain and created a premature stop codon, leading to protein instability and impaired interaction with BRCA1 . Further research using biotin-conjugated antibodies can help identify additional mutations and their functional consequences in breast cancer development.

What experimental design considerations are important when using BRIP1 antibodies in Fanconi anemia pathway research?

When investigating the Fanconi anemia pathway using BRIP1 antibodies:

• Temporal Analysis of Pathway Activation:

  • BRIP1 acts late in the Fanconi anemia pathway, after FANCD2 ubiquitination

  • Sequential immunoprecipitation experiments can capture the temporality of protein complex formation

• Subcellular Localization Studies:

  • BRIP1 translocates to nuclear foci upon DNA damage

  • Biotin-conjugated antibodies in immunofluorescence can track this movement with high sensitivity

• Protein-Protein Interaction Network:

  • Pull-down assays using biotin-conjugated BRIP1 antibodies can identify novel interacting partners

  • Validation through reciprocal co-immunoprecipitation confirms specific interactions

• Functional Recovery Experiments:

  • Complementation studies in BRIP1-deficient cells

  • Quantitative assessment of DNA repair efficiency using immunofluorescence markers

The data generated can be analyzed using pathway mapping tools to position BRIP1 accurately within the Fanconi anemia pathway and its relationship to DNA repair mechanisms, particularly in resolving DNA double-strand breaks through homologous recombination .

What emerging applications of biotin-conjugated BRIP1 antibodies show promise for advancing DNA repair research?

Several emerging applications of biotin-conjugated BRIP1 antibodies show significant promise:

• Single-Cell Proteomics:

  • Integration with mass cytometry (CyTOF) using biotin-conjugated antibodies for high-dimensional analysis

  • Correlation of BRIP1 expression with other DNA repair proteins at single-cell resolution

• In Vivo Imaging:

  • Development of biotin-conjugated antibody fragments for improved tissue penetration

  • Combination with near-infrared fluorophore-labeled streptavidin for in vivo detection of BRIP1 in xenograft models

• Proximity Ligation Assays:

  • Investigation of transient protein-protein interactions in intact cells

  • Spatial resolution of BRIP1-containing complexes during DNA damage response

• CRISPR-Cas9 Screening:

  • Biotin-conjugated antibodies for high-throughput detection of BRIP1 levels following genetic perturbation

  • Identification of synthetic lethal interactions with BRIP1 mutations