BRIP1 Antibody, HRP conjugated

What is BRIP1 and why is it important in research?

BRIP1 (also known as BACH1, FANCJ) is a DNA helicase that interacts with BRCA1 and plays a crucial role in DNA damage repair pathways. In humans, BRIP1 is a 1249 amino acid protein with a molecular weight of approximately 140.9 kDa that localizes to both the nucleus and cytoplasm . It belongs to the DEAD box helicase family (DEAH subfamily) and contains a helicase ATP-binding domain . BRIP1 is of particular research interest due to its involvement in cancer biology, as it has been identified as a potential low-penetrance breast cancer predisposing gene . Its function in resolving secondary structures of guanine-rich DNA during lagging-strand DNA synthesis makes it critical for maintaining genomic stability .

What are the key characteristics of HRP-conjugated BRIP1 antibodies?

HRP-conjugated BRIP1 antibodies combine the specificity of BRIP1 detection with the signal amplification capabilities of horseradish peroxidase (HRP). These conjugated antibodies typically come in liquid form and contain preservatives such as 0.03% Proclin 300 in buffer solutions (50% Glycerol, 0.01M PBS, pH 7.4) . They are purified using methods like Protein G purification to achieve >95% purity . The conjugation to HRP eliminates the need for secondary antibody incubation, streamlining experimental workflows and potentially reducing background signal in certain applications.

What are the common synonyms and alternative names for BRIP1 in the scientific literature?

When searching literature or antibody databases, researchers should be aware of BRIP1's multiple nomenclatures:

• BRCA1 interacting protein C-terminal helicase 1 (full name)

• BRCA1-associated C-terminal helicase 1 (BACH1)

• Fanconi anemia group J protein (FACJ)

• ATP-dependent RNA helicase BRIP1

• BRCA1-binding helicase-like protein BACH1

This diversity in naming can complicate literature searches, so comprehensive search strategies should include all relevant terms.

What are the validated applications for HRP-conjugated BRIP1 antibodies?

How should optimal antibody dilutions be determined for different experimental applications?

For HRP-conjugated BRIP1 antibodies, optimal dilution determination is application-dependent. For ELISA, a titration experiment should be performed with serial dilutions (typically starting at 1:1000 and proceeding to 1:64,000) to generate a standard curve. For reference, unconjugated BRIP1 antibodies are typically used at dilutions of 1:500-1:2000 for Western blotting .

A systematic titration approach includes:

• Preparing serial dilutions of the antibody

• Running identical samples with different antibody dilutions

• Analyzing signal-to-noise ratio for each dilution

• Selecting the dilution that provides optimal specific signal with minimal background

It's important to note that sample type and experimental conditions can significantly impact optimal dilution, making it necessary to optimize for each specific experimental setup.

What controls should be included when working with BRIP1 antibodies in research applications?

Rigorous experimental design requires appropriate controls:

• Positive controls: Cell lines with confirmed BRIP1 expression such as HeLa or MCF-7 cells

• Negative controls:

  • Primary antibody omission control

  • Isotype control (rabbit IgG at equivalent concentration)

  • BRIP1-knockdown or knockout samples when available

• Loading controls: For Western blot applications, use housekeeping proteins (β-actin, GAPDH) to normalize BRIP1 expression

• Specificity controls: Pre-absorption with immunizing peptide to verify signal specificity

These controls help distinguish specific BRIP1 detection from potential artifacts or non-specific binding.

What are the optimal storage conditions for maintaining HRP-conjugated BRIP1 antibody activity?

HRP-conjugated BRIP1 antibodies require careful handling to maintain enzymatic activity and binding specificity:

• Store at -20°C or -80°C as recommended by manufacturers

• Avoid repeated freeze-thaw cycles that may degrade both the antibody and HRP enzyme activity

• For antibodies in 50% glycerol, aliquoting may be unnecessary for -20°C storage

• Once thawed for use, store temporarily at 4°C and use within 1-2 weeks

• Protect from light exposure, as HRP is sensitive to photobleaching

Following these storage guidelines helps preserve antibody performance throughout the expected shelf life of one year after shipment .

What are common troubleshooting approaches for weak or absent signal when using HRP-conjugated BRIP1 antibodies?

When encountering signal problems with HRP-conjugated BRIP1 antibodies, consider:

• Antibody activity:

  • Verify HRP enzyme activity using a simple dot blot with substrate

  • Check antibody expiration date and storage history

• Antigen accessibility:

  • For Western blots, ensure complete protein denaturation

  • For ELISA, optimize coating conditions and blocking buffers

  • Consider alternative epitope antibodies if the target region may be masked

• Detection system:

  • Verify substrate freshness and proper development time

  • Increase exposure time for Western blots or chemiluminescent detection

  • Use signal enhancement systems if necessary

• Protein expression levels:

  • BRIP1 expression varies between tissues, with highest levels reported in testis

  • Consider enrichment steps for low-abundance samples

• Technical parameters:

  • Decrease antibody dilution (use more concentrated antibody)

  • Extend incubation time at 4°C (overnight incubation may improve signal)

  • Verify buffer compatibility with HRP activity

How can researchers differentiate between BRIP1 isoforms when using antibodies?

BRIP1 has two reported isoforms from alternative splicing . To differentiate between them:

• Epitope location awareness: Determine if the antibody targets the N-terminal or C-terminal region . N-terminal targeting antibodies may detect both isoforms, while C-terminal antibodies may be isoform-specific depending on the splice variants.

• Western blot analysis: Carefully analyze molecular weight patterns:

  • Full-length BRIP1: 140-150 kDa

  • Isoforms may appear as distinct bands with different molecular weights

• Isoform-specific controls: When available, use recombinant proteins of specific isoforms as positive controls.

• Complementary techniques: Consider combining antibody detection with RT-PCR using isoform-specific primers to correlate protein and mRNA expression patterns.

How can HRP-conjugated BRIP1 antibodies be utilized to study BRIP1-BRCA1 interactions in DNA damage response?

Investigating BRIP1-BRCA1 interactions requires sophisticated approaches:

• Proximity ligation assays (PLA): HRP-conjugated BRIP1 antibodies can be paired with BRCA1 antibodies to detect protein-protein interactions at DNA damage sites with spatial resolution.

• Chromatin immunoprecipitation (ChIP): HRP-conjugated antibodies can be adapted for ChIP experiments to study BRIP1 recruitment to DNA damage sites.

• Co-localization studies: Using BRIP1 antibodies in combination with BRCA1 detection to visualize their co-localization at DNA repair foci following various DNA damaging treatments .

• Functional analysis: Monitor BRIP1-BRCA1 interactions before and after DNA damage induction using various genotoxic agents to understand the kinetics of complex formation.

These approaches can help elucidate how BRIP1 contributes to DNA repair function through its interaction with BRCA1, which is critical for understanding its role in genomic stability .

What methodological approaches can be used to study BRIP1 post-translational modifications?

BRIP1 undergoes several post-translational modifications including acetylation and phosphorylation . To study these:

• Phospho-specific antibodies: Use antibodies targeting specific phosphorylation sites, such as Ser990 , in combination with:

  • Phosphatase treatments as controls

  • Kinase inhibitors to identify regulatory pathways

• IP-Western analysis:

  • Immunoprecipitate total BRIP1 first

  • Then probe with modification-specific antibodies (anti-acetyl lysine, anti-phospho serine/threonine)

• Mass spectrometry approaches:

  • Enrichment of BRIP1 by immunoprecipitation

  • Tryptic digestion followed by LC-MS/MS analysis

  • Comparison of modification patterns under different cellular conditions

• Functional correlation:

  • Correlate post-translational modification status with helicase activity

  • Analyze how modifications affect BRCA1 binding capabilities

How can researchers optimize BRIP1 detection in cellular hypoxia response studies?

BRIP1 is involved in cellular response to hypoxia . For studying this connection:

• Hypoxia induction protocols:

  • Chemical mimetics (CoCl₂, DMOG) vs. true hypoxic chambers

  • Time-course experiments to capture dynamic regulation

• Cell fractionation optimization:

  • Nuclear vs. cytoplasmic fractionation to track BRIP1 translocation

  • Chromatin-bound vs. soluble nuclear fractions to assess functional engagement

• Co-detection strategies:

  • Simultaneous probing for HIF-1α and BRIP1

  • Correlation with hypoxia-responsive genes

• Signal amplification:

  • Tyramide signal amplification (TSA) for immunofluorescence applications

  • Enhanced chemiluminescence substrates for Western blot detection

• Normalization considerations:

  • Use of hypoxia-stable reference genes/proteins rather than traditional housekeeping genes

  • Relative quantification against total protein staining methods

How should researchers interpret variations in BRIP1 expression patterns across different cell types?

BRIP1 is widely expressed across many tissue types with highest levels in testis . When analyzing expression data:

• Baseline expression establishment:

  • Create a panel of normal tissues/cells for comparative analysis

  • Consider developmental stage and cell cycle phase influences

• Quantification methods:

  • Use densitometry for Western blot quantification

  • Employ digital image analysis for immunohistochemistry/immunofluorescence

  • Always normalize to appropriate loading controls

• Analysis of subcellular localization:

  • Nuclear vs. cytoplasmic distribution varies by cell type

  • Changes in localization may indicate functional shifts

• Correlation with functional state:

  • DNA repair capacity

  • Cell cycle stage

  • Differentiation status

Understanding these variations is crucial for correctly interpreting experimental results across different model systems.

What considerations are important when analyzing BRIP1 expression in cancer samples?

BRIP1 has been implicated as a breast cancer susceptibility gene . When studying cancer samples:

• Heterogeneity considerations:

  • Tumor vs. stroma expression patterns

  • Intratumoral heterogeneity requiring multiple sampling

  • Cancer subtype-specific expression profiles

• Mutation status correlation:

  • Sequence BRIP1 gene for potential mutations

  • Correlate expression with mutation status

  • Assess impact on protein function and stability

• Prognostic value assessment:

  • Correlation with clinical outcomes

  • Multivariate analysis with established biomarkers

  • Survival analysis methods (Kaplan-Meier, Cox regression)

• Therapy response prediction:

  • Changes in expression following DNA-damaging treatments

  • Correlation with resistance to platinum drugs or PARP inhibitors

  • Potential as a companion diagnostic marker

These considerations help contextualize BRIP1 expression data within the broader clinical and molecular landscape of cancer research.