FAM175A Antibody

What is FAM175A and why is it important in research?

FAM175A (Family with sequence similarity 175 member A) is a member of the DNA repair protein family that contributes significantly to BRCA1-dependent DNA damage response pathways. The protein has a calculated molecular weight of 47 kDa, though it is typically observed at approximately 46 kDa in experimental conditions . FAM175A is also known by several alternative names including ABRAXAS1, CCDC98, and ABRA1, which reflects its discovery by different research groups investigating various aspects of DNA repair mechanisms . The protein's importance stems from its role in genome stability maintenance, with research showing that germline variants have been identified in high-risk breast cancer families, particularly in Northern Finnish populations . FAM175A forms a complex with BRCA1 that is essential for proper DNA damage repair signaling, making it a critical research target for understanding cancer predisposition and potential therapeutic vulnerabilities in DNA repair pathways . Studies of FAM175A function provide insights into fundamental cellular processes of DNA damage recognition and repair that are frequently dysregulated in cancer development.

How do I select the appropriate FAM175A antibody for my specific experimental applications?

Selection of an appropriate FAM175A antibody should begin with a careful assessment of your experimental requirements, including the application type, sample species, and desired sensitivity. For Western blot applications, polyclonal antibodies like the 14366-1-AP (Proteintech) demonstrate broad reactivity across multiple human cell lines including HEK-293T, MCF-7, Hela, Jurkat cells, and human testis tissue, with recommended dilutions ranging from 1:500 to 1:3000 . For immunofluorescence or immunocytochemistry, this same antibody has been validated in HeLa and MCF-7 cells at dilutions of 1:50 to 1:500 . When cross-species reactivity is required, the CUSABIO CSB-PA008138GA01HU antibody might be preferred as it demonstrates reactivity with human, mouse, and rat samples . For applications requiring higher specificity, consider the mouse monoclonal antibody (MAB8508, clone ABRA1-01) from Abnova, which is specifically targeted to the N-terminal region (amino acids 1-313) of human FAM175A and may be particularly useful for flow cytometry applications . Always verify the validation data for the specific application and examine literature where these antibodies have been successfully employed in similar experimental contexts. Additionally, consider the recognition epitope in relation to your research question—for instance, if studying a specific domain or post-translational modification of FAM175A.

What controls should I include when validating a FAM175A antibody for my research?

Proper validation of FAM175A antibodies requires a comprehensive set of controls to ensure specificity and reliability of experimental results. First, include a positive control consisting of cells or tissues known to express FAM175A, such as HEK-293T, MCF-7, HeLa, or Jurkat cells, which have been validated for FAM175A expression with antibodies like 14366-1-AP . A negative control should be implemented through siRNA or CRISPR-mediated knockdown/knockout of FAM175A to confirm that the observed signal is specifically reduced or eliminated with target depletion. For further validation, consider using cells from FAM175A-mutated samples such as those carrying the c.1106dup or c.577C>T mutations that have been described in breast cancer studies, which can display altered expression patterns . When performing immunofluorescence, include co-localization studies with known interacting partners like BRCA1, as their co-localization increases significantly (approximately five-fold) following irradiation-induced DNA damage . Additionally, implement technical controls including primary antibody omission and isotype controls (rabbit IgG for polyclonal antibodies like 14366-1-AP and CSB-PA008138GA01HU, or mouse IgG for monoclonal antibodies like MAB8508) . These validation steps ensure that your experimental findings with FAM175A antibodies are both specific and reproducible.

How can I optimize Western blot protocols for FAM175A detection?

Optimizing Western blot protocols for FAM175A detection requires attention to several critical parameters to ensure specific and sensitive results. Begin with proper sample preparation by extracting proteins in a buffer containing protease inhibitors to prevent degradation of the 46-47 kDa FAM175A protein . For cell lysis, RIPA buffer supplemented with phosphatase inhibitors is recommended, particularly when studying FAM175A phosphorylation states that may occur following DNA damage. Use 20-40 μg of total protein per lane and separate on an 8-10% SDS-PAGE gel to achieve optimal resolution around the 46 kDa range where FAM175A is typically observed . For transfer, a PVDF membrane often provides better results than nitrocellulose for this protein. When blocking, 5% non-fat dry milk in TBST is generally effective, though for phospho-specific detection, 5% BSA may yield superior results. For primary antibody incubation with polyclonal antibodies like 14366-1-AP, begin with a 1:1000 dilution in appropriate buffer overnight at 4°C, recognizing that the recommended range extends from 1:500 to 1:3000 . Following thorough washing steps (4-5 times with TBST), apply HRP-conjugated secondary antibody at 1:5000 dilution. For enhanced sensitivity, particularly when examining FAM175A in samples with low expression levels, consider using enhanced chemiluminescence detection systems or fluorescently-labeled secondary antibodies with digital imaging systems that provide greater linear dynamic range for quantification.

What protocols are most effective for immunofluorescence studies of FAM175A in relation to DNA damage response?

Effective immunofluorescence protocols for studying FAM175A in DNA damage response contexts require careful optimization of fixation, permeabilization, and antibody incubation steps. Begin by growing cells on coverslips coated with poly-L-lysine to ensure proper adherence and distribution. For DNA damage induction, treat cells with ionizing radiation (typical doses range from 2-10 Gy) or DNA damaging agents like etoposide (10-20 μM) for 1-6 hours before fixation . Fix cells with 4% paraformaldehyde for 15 minutes at room temperature, followed by permeabilization with 0.2% Triton X-100 for 10 minutes. Blocking with 5% normal serum (goat or donkey, depending on secondary antibody species) containing 0.1% Triton X-100 for 1 hour helps minimize non-specific binding. For primary antibody incubation, use FAM175A antibody at dilutions of 1:50 to 1:500 (starting at 1:100 is recommended) in blocking buffer overnight at 4°C . To effectively study FAM175A's role in DNA damage response, co-staining with markers such as γH2AX (Ser139), 53BP1, or BRCA1 is highly informative, as increased co-localization of FAM175A with BRCA1 foci (up to fivefold) following irradiation has been documented . Apply appropriate fluorescent secondary antibodies at 1:500 dilution for 1 hour at room temperature in the dark. Counterstain nuclei with DAPI and mount slides with anti-fade mounting medium. For quantitative analysis, collect Z-stack images using confocal microscopy and analyze foci numbers, sizes, and co-localization patterns using software like ImageJ with the JACoP plugin.

How can I effectively use FAM175A antibodies in flow cytometry applications?

Optimizing flow cytometry protocols for FAM175A detection requires careful preparation to preserve protein epitopes while ensuring adequate permeabilization for antibody access to this nuclear protein. Begin with appropriate cell fixation using 2-4% paraformaldehyde for 15 minutes at room temperature, followed by permeabilization with 0.1-0.3% Triton X-100 or commercially available permeabilization buffers specifically designed for nuclear proteins. When using monoclonal antibodies like MAB8508 (clone ABRA1-01), a concentration of 1-2 μg/mL is recommended as a starting point for flow cytometry applications . For polyclonal antibodies, titration experiments starting from 1:100 to 1:500 dilutions are advisable to determine optimal signal-to-noise ratios. Include appropriate isotype controls (mouse IgG for monoclonal antibodies like MAB8508 or rabbit IgG for polyclonal antibodies) at equivalent concentrations to assess non-specific binding . When studying FAM175A in the context of the cell cycle or DNA damage response, consider dual staining with propidium iodide or DAPI for DNA content analysis, or with γH2AX antibodies to correlate FAM175A expression with DNA damage levels. For cell sorting applications where FAM175A expression levels are used as selection criteria, implement stringent gating strategies based on negative and positive controls to ensure high purity of sorted populations. When analyzing data, use fluorescence minus one (FMO) controls to accurately set gates and consider using median fluorescence intensity rather than mean values to account for potential population heterogeneity.

How can I utilize FAM175A antibodies to investigate its interaction with BRCA1 and the DNA damage response pathway?

To effectively investigate FAM175A interactions with BRCA1 and its role in DNA damage response pathways, researchers can employ several advanced antibody-based techniques. Co-immunoprecipitation (Co-IP) represents a powerful approach, where cell lysates from normal or DNA damage-treated samples are immunoprecipitated with FAM175A antibodies (such as 14366-1-AP or CSB-PA008138GA01HU), followed by immunoblotting for BRCA1 and other potential interacting partners . Proximity ligation assays (PLA) offer an alternative in situ approach to visualize and quantify protein-protein interactions between FAM175A and BRCA1 at the single-molecule level within cells. For studying recruitment dynamics to DNA damage sites, combine micro-irradiation techniques using UV or laser systems with live-cell imaging of cells expressing fluorescently-tagged FAM175A, validating observations with fixed-cell immunofluorescence using FAM175A antibodies . Chromatin immunoprecipitation (ChIP) with FAM175A antibodies can reveal its association with chromatin at specific genomic loci during DNA repair processes. To investigate the functional consequences of FAM175A mutations (such as c.1106dup or c.577C>T) on BRCA1 interaction, implement co-localization studies comparing wild-type versus mutant cells, as research has shown that these mutations can significantly alter BRCA1 foci formation and co-localization patterns . For a comprehensive understanding of the DNA damage response network centered around FAM175A, consider combining antibody-based proximity-dependent biotinylation (BioID) approaches with mass spectrometry to identify the complete interactome under various damage conditions.

What approaches can help distinguish between specific and non-specific signals when using FAM175A antibodies in complex biological samples?

Distinguishing specific from non-specific signals when using FAM175A antibodies in complex biological samples requires implementation of comprehensive validation strategies and appropriate controls. Begin with genetic validation approaches by comparing signals between wild-type samples and those where FAM175A has been depleted through siRNA knockdown, shRNA, or CRISPR-Cas9 knockout; a true specific signal should be substantially reduced or eliminated in FAM175A-depleted samples. Peptide competition assays provide another validation method, where pre-incubation of the antibody with excess immunizing peptide should block specific binding to FAM175A in subsequent applications. When working with tissue samples, consider dual-staining approaches using two different FAM175A antibodies targeting distinct epitopes, such as combining a monoclonal antibody targeting the N-terminal region (like MAB8508) with a polyclonal antibody; co-localization of signals strongly supports specificity . Technical controls should include primary antibody omission, isotype controls (rabbit IgG for polyclonal antibodies or mouse IgG for monoclonal antibodies like MAB8508), and concentration-matched non-immune serum controls . For Western blots, the molecular weight serves as an important criterion, with FAM175A appearing at approximately 46 kDa as observed in validated experiments . In immunofluorescence studies, specific FAM175A signals should display the expected subcellular localization pattern and respond appropriately to experimental manipulations such as DNA damage induction, which causes distinct focal accumulation . For advanced validation, consider orthogonal approaches such as mass spectrometry analysis of immunoprecipitated proteins to confirm the identity of the detected bands or spots, particularly in post-translational modification studies.

How can FAM175A antibodies be used to investigate its potential roles beyond DNA repair?

Investigating FAM175A functions beyond DNA repair requires innovative applications of antibodies to explore its potential involvement in additional cellular processes. Researchers can implement co-immunoprecipitation with FAM175A antibodies followed by mass spectrometry to identify novel interaction partners outside the canonical BRCA1-A complex, potentially revealing unexpected functional relationships . For studying potential roles in transcriptional regulation, chromatin immunoprecipitation (ChIP) with FAM175A antibodies followed by sequencing (ChIP-seq) can map its genomic binding sites, with differential binding analysis under various cellular conditions providing insights into context-dependent functions. The observed association between FAM175A and age at natural menopause suggests potential roles in reproductive biology or aging that could be explored through immunohistochemistry in reproductive tissues across age ranges or in model systems of cellular senescence . To investigate possible cytoplasmic functions, implement subcellular fractionation followed by Western blotting with antibodies like 14366-1-AP, which has been validated across multiple cell types . Proximity-based labeling approaches such as BioID or APEX2 fused to FAM175A, validated by antibody detection, can reveal spatial proteomics information about FAM175A neighborhoods in different cellular compartments. For studying potential roles in cell cycle regulation, combine flow cytometry for DNA content with FAM175A immunostaining using antibodies like MAB8508, which is recommended for flow cytometry applications . Given the connections between DNA repair deficiencies and immune responses, investigate potential links between FAM175A and inflammatory signaling through co-immunostaining with markers of inflammation or stress responses in cellular and tissue models.

What methodological approaches can be used to study post-translational modifications of FAM175A?

Studying post-translational modifications (PTMs) of FAM175A requires specialized methodological approaches that extend beyond standard antibody applications. Researchers should begin with phospho-specific antibody development or acquisition, targeting known or predicted phosphorylation sites on FAM175A that may be regulated during DNA damage responses or cell cycle progression. For comprehensive PTM profiling, implement immunoprecipitation with validated FAM175A antibodies like 14366-1-AP or CSB-PA008138GA01HU followed by mass spectrometry analysis, ideally comparing samples from different cellular conditions such as before and after DNA damage induction . Phos-tag SDS-PAGE represents another valuable technique, where phosphorylated forms of FAM175A exhibit reduced mobility compared to non-phosphorylated forms, allowing separation and detection with standard FAM175A antibodies. To study ubiquitination or SUMOylation, use denaturing conditions during immunoprecipitation to disrupt protein-protein interactions, followed by immunoblotting with anti-ubiquitin or anti-SUMO antibodies. For functional studies of specific modifications, combine site-directed mutagenesis of PTM sites with rescue experiments in FAM175A-depleted backgrounds, validating expression and localization patterns with immunofluorescence using antibodies like 14366-1-AP, which has been validated for this application . Proximity ligation assays (PLA) with pairs of antibodies targeting FAM175A and specific modifications (such as phospho-epitopes or ubiquitin) can visualize modified protein populations in situ. To connect PTMs with functional outcomes, implement immunofluorescence co-localization studies examining how specific mutations affecting modification sites influence interactions with DNA damage response proteins like BRCA1, which normally shows significantly increased co-localization with FAM175A following DNA damage .

How might FAM175A antibodies contribute to understanding potential therapeutic vulnerabilities in cancer?

FAM175A antibodies can significantly advance our understanding of therapeutic vulnerabilities in cancer through several sophisticated research approaches. Immunohistochemistry profiling of FAM175A expression and localization patterns across diverse tumor types can identify cancer subtypes with altered FAM175A status that might exhibit specific therapeutic sensitivities. Functional studies correlating FAM175A expression or mutation status with response to PARP inhibitors like olaparib or 1,5-isoquinolinediol (IQD) can be conducted, as research suggests connections between BRCA1 pathway alterations and PARP inhibitor sensitivity, though studies with heterozygous ABRAXAS1 mutations did not show statistically significant changes in sensitivity . For investigating synthetic lethal interactions, researchers can implement siRNA or CRISPR screens in FAM175A-deficient versus proficient backgrounds, using antibody-based detection methods to validate knockdown efficiency and correlate with phenotypic outcomes. Combination therapy approaches targeting FAM175A-deficient tumors can be explored by examining how FAM175A status influences response to standard chemotherapies or emerging targeted agents using cell viability assays and immunofluorescence to monitor DNA damage accumulation. Recent research demonstrated that FAM175A mutations impact DNA repair pathway choice, with cells carrying the c.577C>T mutation showing threefold increased frequencies of both homologous recombination and non-homologous end joining, while both c.1106dup and c.577C>T mutations led to twofold enhanced single-strand annealing frequencies . These pathway alterations could potentially be exploited therapeutically. Additionally, the finding that PI3K-AKT or RTK-MAPK pathway activation strongly indicates poor clinical outcomes in some cancers suggests potential combination strategies targeting these pathways alongside DNA repair inhibitors in tumors with FAM175A deficiencies . Biomarker development studies using FAM175A antibodies could facilitate patient stratification for clinical trials testing such therapeutic approaches.