RNF19A Antibody, Biotin conjugated

What is RNF19A and why is it important in cancer research?

RNF19A is a ring between ring fingers (RBR) family E3 ligase that regulates DNA repair pathways by interacting with and ubiquitinating BARD1, which leads to the dissociation of the BRCA1-BARD1 complex. This dissociation compromises homologous recombination (HR) repair and increases cancer cell sensitivity to PARP inhibitors (PARPi). RNF19A has been implicated in NF-κB signaling, fertilization, and neuroinflammation, and its mRNA is amplified in the blood of prostate cancer patients, suggesting broader implications in cancer biology beyond DNA repair .

How does biotin conjugation enhance RNF19A antibody applications?

Biotin conjugation leverages one of nature's strongest non-covalent interactions—the avidin-biotin binding system—to enhance detection sensitivity and versatility. The biotin-conjugated RNF19A antibody can interact with avidin, streptavidin, or neutravidin systems, allowing for signal amplification, efficient operation in detection systems, and high stability under varying experimental conditions. This conjugation enables researchers to use highly diluted primary antibodies while maintaining robust detection, particularly useful when working with low-abundance proteins or when multiple detection steps are required .

What are the primary research applications for RNF19A biotin-conjugated antibodies?

The primary applications include:

• Immunoprecipitation to study RNF19A-BARD1 interactions

• Immunofluorescence microscopy to visualize subcellular localization

• Flow cytometry for quantitative analysis of expression levels

• Western blotting for protein expression analysis

• ChIP assays to study RNF19A recruitment to DNA damage sites

• Pull-down assays leveraging the avidin-biotin system for complex purification

• Immunohistochemistry for tissue expression pattern analysis

What experimental controls should be included when using RNF19A biotin-conjugated antibodies?

Essential controls include:

These controls ensure reliable interpretation of results, particularly when studying RNF19A's complex interactions with the BRCA1-BARD1 system .

How can RNF19A biotin-conjugated antibodies be used to study protein-protein interactions?

For studying RNF19A interactions with proteins like BARD1, researchers can implement a sequential immunoprecipitation strategy. First, perform a standard immunoprecipitation using the biotin-conjugated RNF19A antibody with streptavidin beads. After elution, conduct a second immunoprecipitation with an antibody against the potential interacting protein (e.g., BARD1). This approach, when combined with mass spectrometry analysis, can identify novel RNF19A binding partners beyond those already known, such as BARD1.

Research has demonstrated that RNF19A specifically interacts with BARD1 through its RING1 domain, while BARD1 interacts with RNF19A through its RING domain. These interactions can be confirmed through GST pull-down assays with purified proteins, where GST-RNF19A directly interacts with His-BARD1, an interaction disrupted by deletion of RNF19A's RING1 domain .

What methods can resolve contradictory data regarding RNF19A's role in DNA damage response?

When confronting contradictory findings regarding RNF19A's role in DNA damage response, implement a multi-faceted validation approach:

• Functional complementation assays: In RNF19A-deficient cells, reintroduce either wild-type RNF19A or catalytically inactive mutants (C316A) to determine whether the E3 ligase activity is essential for the observed phenotypes.

• Domain-specific analysis: Express truncated versions of RNF19A (RING1 deletion, IBR deletion, or RING2 deletion) to determine which domains are critical for specific functions.

• Temporal dynamics assessment: Perform time-course experiments after DNA damage induction to observe how RNF19A affects the recruitment kinetics of repair factors like BRCA1, BARD1, RPA32, and RAD51.

• Cell cycle synchronization: As DNA repair pathway choice is cell-cycle dependent, synchronize cells in different cell cycle phases to determine if RNF19A's effects are cell cycle-specific.

Published research has shown that wild-type RNF19A, but not the catalytically inactive mutant, can reverse increased HR efficiency and re-sensitize cells to PARPi when introduced into RNF19A-deficient cells, indicating that the catalytic activity is essential for its function in HR regulation .

How can researchers validate the specificity of ubiquitination patterns mediated by RNF19A?

To validate RNF19A-mediated ubiquitination patterns:

• In vivo ubiquitination assays: Express tagged versions of ubiquitin (HA-Ub or His-Ub) along with RNF19A (wild-type or catalytically inactive) and potential substrates like BARD1. After immunoprecipitation of the substrate, detect ubiquitination using antibodies against the ubiquitin tag.

• Linkage-specific ubiquitin antibodies: Use antibodies specific for different ubiquitin linkages (K48, K63, etc.) to determine the type of chains formed by RNF19A. Research has shown that RNF19A primarily promotes K63-linked ubiquitination of BARD1 .

• In vitro ubiquitination assays: Use purified components (E1, E2, RNF19A, and BARD1) to reconstitute the ubiquitination reaction in a test tube. This approach can directly demonstrate RNF19A's E3 ligase activity toward BARD1 without cellular confounding factors.

• Mass spectrometry analysis: Identify specific lysine residues on BARD1 that are ubiquitinated by RNF19A through mass spectrometry of purified ubiquitinated BARD1.

Studies have confirmed that RNF19A promotes ubiquitination of BARD1 through K63-linked ubiquitin chains, which affects the interaction between BRCA1 and BARD1 rather than protein stability .

What experimental approaches can determine if RNF19A biotin-conjugated antibodies affect target protein function?

To assess whether biotin-conjugated RNF19A antibodies affect the target protein's function:

• Functional comparison: Compare cellular outcomes (e.g., HR efficiency, PARPi sensitivity) when using different antibody formats (unconjugated, biotin-conjugated, directly labeled).

• Epitope mapping: Determine the epitope recognized by the antibody and assess whether it overlaps with functional domains of RNF19A (RING1, IBR, RING2). Antibodies targeting the RING1 domain might interfere with BARD1 binding.

• In vitro activity assays: Test whether the presence of the antibody affects RNF19A's ubiquitination activity in reconstituted in vitro systems.

• Live-cell imaging: Use cell-permeable biotin-conjugated antibody fragments to observe whether binding affects RNF19A's subcellular localization or recruitment to DNA damage sites.

• Competition assays: Determine if the antibody competes with natural binding partners by performing immunoprecipitation in the presence and absence of the antibody.

These approaches help ensure that experimental observations reflect genuine biological phenomena rather than artifacts introduced by the biotin-conjugated antibody .

How should researchers optimize signal-to-noise ratio when using RNF19A biotin-conjugated antibodies?

To optimize signal-to-noise ratio with biotin-conjugated RNF19A antibodies:

• Endogenous biotin blocking: Pre-treat samples with unconjugated avidin/streptavidin to block endogenous biotin, which is abundant in mitochondria and can contribute to background signal.

• Titration optimization: Perform systematic titration experiments to determine the minimum effective concentration of biotin-conjugated antibody that provides specific signal.

• Sequential detection systems: For immunofluorescence or IHC, employ a sequential detection system where the primary biotin-conjugated antibody is detected with fluorophore-conjugated streptavidin, followed by amplification steps if necessary.

• Blocking optimization: Test different blocking solutions (BSA, normal serum, commercial blockers) to minimize non-specific binding of the detection reagent.

• Avidin derivative selection: Compare streptavidin, neutravidin, and avidin for detection, as they differ in non-specific binding properties. Neutravidin typically provides lower background due to reduced charge-based interactions .

What strategies help distinguish between direct and indirect effects of RNF19A on the BRCA1-BARD1 complex?

To distinguish direct from indirect effects:

• Reconstituted systems: Use purified components (RNF19A, BARD1, BRCA1) to demonstrate direct interactions and modifications in vitro. Research has shown that GST-RNF19A directly interacts with His-BARD1 in GST pull-down assays .

• Structure-function mutations: Introduce specific mutations in key domains of RNF19A (RING1, RING2) to disrupt particular functions while preserving others. The RNF19A RING1 domain has been shown to be required for BARD1 interaction .

• Proximity ligation assays: Visualize direct protein-protein interactions in situ with single-molecule resolution to determine whether RNF19A directly associates with BARD1 or BRCA1.

• Sequential ChIP experiments: Determine co-occupancy of RNF19A with BRCA1/BARD1 at sites of DNA damage to establish spatial proximity in a functional context.

• Real-time interaction monitoring: Use techniques like biolayer interferometry or surface plasmon resonance with purified proteins to measure direct binding kinetics.

Studies have confirmed that RNF19A interacts with BARD1 but not with BRCA1, suggesting that effects on BRCA1 localization are mediated through BARD1 .

How can researchers effectively use RNF19A biotin-conjugated antibodies in multiplexed detection systems?

For multiplexed detection involving RNF19A biotin-conjugated antibodies:

• Sequential detection protocols: When combining with other antibodies, perform sequential rather than simultaneous detection to prevent cross-reactivity. For example:

  • First round: Detect non-biotinylated antibodies with conventional secondary antibodies

  • Second round: Detect biotin-conjugated RNF19A antibody with streptavidin conjugates

• Spectral separation: Choose detection fluorophores with minimal spectral overlap to enable clear discrimination between signals.

• Orthogonal labeling systems: Combine the biotin-avidin system with other labeling approaches (e.g., click chemistry, HaloTag) for non-overlapping multiplexed detection.

• Tyramide signal amplification: Use biotinyl-tyramide amplification for weakly expressed targets while directly labeling abundant targets.

• Mass cytometry adaptation: For highly multiplexed analyses, consider adapting to mass cytometry (CyTOF) where biotin-conjugated antibodies can be detected with isotope-labeled streptavidin.

This approach allows simultaneous visualization of RNF19A with interacting partners like BARD1, BRCA1, and other DNA repair factors .

What are the best approaches for quantifying RNF19A-mediated ubiquitination using biotin-conjugated antibodies?

For quantifying RNF19A-mediated ubiquitination:

• Tandem ubiquitin binding entities (TUBEs): Use TUBEs to purify ubiquitinated proteins, followed by detection with biotin-conjugated RNF19A antibodies to quantify RNF19A-associated ubiquitinated species.

• Sequential immunoprecipitation: First immunoprecipitate with ubiquitin antibodies, then detect with biotin-conjugated RNF19A antibodies, or vice versa.

• Proximity ligation assay (PLA): Perform PLA between ubiquitin and RNF19A antibodies to visualize and quantify ubiquitination events at the single-molecule level.

• ELISA-based quantification: Develop sandwich ELISA using capture antibodies against the target protein and detection with either ubiquitin antibodies or biotin-conjugated RNF19A antibodies.

• Flow cytometry: For cell-based quantification, use permeabilized cells stained with biotin-conjugated RNF19A antibodies and ubiquitin antibodies for co-expression analysis.

Studies have demonstrated that RNF19A promotes K63-linked ubiquitination of BARD1, which can be detected in both cellular and in vitro systems, and this ubiquitination affects the interaction between BRCA1 and BARD1 .

How can RNF19A biotin-conjugated antibodies be integrated into nanoparticle-based detection systems?

RNF19A biotin-conjugated antibodies can be effectively integrated into nanoparticle-based detection systems through the following approaches:

• Avidin-coated nanoparticles: Functionalize nanoparticles (gold, quantum dots, magnetic) with avidin/streptavidin, which then capture biotin-conjugated RNF19A antibodies. This modular approach allows for rapid adaptation of the detection system.

• Layer-by-layer assembly: Create multilayered detection systems where avidin-biotin interactions form the connections between different functional layers, with RNF19A antibodies serving as the recognition element.

• Multifunctional nanoplatforms: Develop platforms that combine biotin-conjugated RNF19A antibodies with therapeutic agents, allowing simultaneous detection and targeting of cells with altered RNF19A expression.

• Signal amplification strategies: Incorporate enzymatic amplification systems where biotin-conjugated RNF19A antibodies capture targets, followed by detection with enzyme-labeled streptavidin and subsequent signal generation.

• Theranostic applications: Combine imaging capabilities with therapeutic delivery by using avidin-biotin linkages to attach both RNF19A antibodies and drug payloads to nanoparticles.

These approaches leverage the versatility and high binding affinity of the avidin-biotin system to create sophisticated detection platforms with enhanced sensitivity and functionality compared to conventional methods .

What methodologies can assess the impact of RNF19A expression on patient response to PARP inhibitors?

To assess correlations between RNF19A expression and PARP inhibitor response:

• Tissue microarray analysis: Use biotin-conjugated RNF19A antibodies in immunohistochemistry of patient tumor samples arranged in tissue microarrays to quantify expression levels relative to treatment outcomes.

• Ex vivo drug sensitivity testing: Culture patient-derived organoids or tumor explants, assess RNF19A expression with biotin-conjugated antibodies, and correlate with PARP inhibitor sensitivity in culture.

• Circulating tumor cell analysis: Develop microfluidic platforms using biotin-conjugated RNF19A antibodies to isolate and characterize circulating tumor cells before and during PARP inhibitor treatment.

• Multiplex biomarker panels: Create panels that simultaneously assess RNF19A, BRCA1, BARD1, and other HR pathway components to develop comprehensive predictive signatures.

• Functional HR assays: Develop cell-based reporter systems that measure HR efficiency in patient samples and correlate with RNF19A expression levels detected by biotin-conjugated antibodies.

Research has shown that RNF19A suppresses HR by ubiquitinating BARD1, leading to dissociation of the BRCA1-BARD1 complex and increased sensitivity to PARP inhibitors, suggesting RNF19A could serve as a biomarker for PARP inhibitor response .

How can researchers study the dynamic interactions between RNF19A and BARD1 during DNA damage response?

To study dynamic RNF19A-BARD1 interactions during DNA damage:

• Real-time live-cell imaging: Use fluorescently tagged proteins combined with photobleaching techniques (FRAP, FLIP) to monitor the kinetics of RNF19A and BARD1 recruitment to DNA damage sites.

• Temporal ChIP analysis: Perform chromatin immunoprecipitation with biotin-conjugated RNF19A antibodies at different time points after DNA damage induction to track recruitment dynamics.

• Proximity-based labeling: Employ BioID or APEX2 fusion proteins to identify proteins in proximity to RNF19A at different stages of the DNA damage response.

• Single-molecule tracking: Implement super-resolution microscopy with biotin-conjugated antibody fragments to track individual RNF19A molecules during the damage response.

• Synchronized damage induction: Use laser microirradiation or site-specific nucleases combined with time-lapse imaging of fluorescently tagged RNF19A and BARD1 to observe recruitment order and dynamics.

Studies have shown that RNF19A influences the retention of BRCA1/BARD1 at DNA double-strand breaks, affecting downstream recruitment of repair factors like RPA32 and RAD51, which are critical for homologous recombination .

What specialized techniques can reveal the structural changes induced by RNF19A-mediated ubiquitination of BARD1?

To investigate structural changes resulting from RNF19A-mediated ubiquitination:

• Hydrogen-deuterium exchange mass spectrometry: Compare deuterium incorporation patterns between non-ubiquitinated and RNF19A-ubiquitinated BARD1 to identify regions with altered solvent accessibility.

• Single-particle cryo-electron microscopy: Visualize structural conformations of BRCA1-BARD1 complexes with and without RNF19A-mediated ubiquitination.

• FRET-based conformational sensors: Design FRET pairs positioned at strategic locations in BARD1 to detect conformational changes upon ubiquitination.

• Nuclear magnetic resonance spectroscopy: Perform NMR analysis of isotopically labeled BARD1 domains before and after ubiquitination to detect structural perturbations.

• Molecular dynamics simulations: Complement experimental data with computational modeling of how K63-linked ubiquitin chains affect BARD1 structure and BRCA1 binding.

Research has revealed that RNF19A-mediated ubiquitination of BARD1 leads to exposure of a nuclear export sequence that is normally masked by BRCA1 binding, resulting in cytoplasmic relocalization of BARD1 and compromised HR repair .

How might RNF19A biotin-conjugated antibodies be used to develop novel cancer diagnostics?

RNF19A biotin-conjugated antibodies offer several promising avenues for cancer diagnostic development:

• Liquid biopsy applications: Develop microfluidic devices with immobilized streptavidin to capture RNF19A-biotin antibody complexes bound to circulating tumor DNA or exosomes, enabling non-invasive detection of cancer biomarkers.

• Multiplexed tissue imaging: Create multiplexed imaging panels incorporating biotin-conjugated RNF19A antibodies alongside markers for DNA damage, cell cycle, and apoptosis to comprehensively profile tumor tissue and predict therapy response.

• Point-of-care testing: Engineer lateral flow or microarray platforms using the biotin-streptavidin system for rapid assessment of RNF19A expression or activity in clinical samples.

• Companion diagnostics: Develop standardized assays measuring RNF19A expression or activity to identify patients likely to respond to PARP inhibitors or other targeted therapies.

• Functional pathway diagnostics: Create cell-based reporter systems that integrate RNF19A detection with functional assessment of HR pathway activity to provide more accurate prediction of therapeutic responses.

Given RNF19A's role in regulating the BRCA1-BARD1 complex and DNA repair pathways, such diagnostics could help identify patients with dysfunctional HR who might benefit from PARP inhibitors, even in the absence of BRCA mutations .

What research gaps remain in understanding RNF19A's role across different cancer types?

Despite progress, significant research gaps remain regarding RNF19A:

• Cancer-type specificity: Most studies have focused on breast cancer, leaving RNF19A's role in other cancers largely unexplored despite evidence of its aberrant expression in prostate cancer and cancer-associated fibroblasts .

• Regulatory mechanisms: The upstream regulation of RNF19A expression and activity remains poorly understood, including potential post-translational modifications that might control its function.

• Additional substrates: While BARD1 has been identified as a substrate, the complete repertoire of RNF19A targets in different cellular contexts is unknown.

• Non-canonical functions: Potential roles beyond ubiquitination have not been extensively investigated.

• Prognostic value: The correlation between RNF19A expression/activity and patient outcomes across cancer types needs systematic evaluation.

• Therapeutic targeting: The feasibility of directly targeting RNF19A as a therapeutic approach has not been thoroughly explored.

Addressing these gaps will require comprehensive profiling of RNF19A across cancer types using biotin-conjugated antibodies for detection, combined with functional studies to elucidate context-specific roles .

How can RNF19A biotin-conjugated antibodies contribute to understanding therapy resistance mechanisms?

To investigate therapy resistance mechanisms:

• Sequential tumor biopsies: Analyze RNF19A expression and localization in matched pre-treatment and post-resistance tumor samples using biotin-conjugated antibodies.

• Resistance model characterization: Compare RNF19A-mediated BARD1 ubiquitination patterns between therapy-sensitive and resistant cell lines using immunoprecipitation with biotin-conjugated antibodies.

• Functional genomics screening: Combine CRISPR screening with biotin-conjugated RNF19A antibody-based detection to identify genes that modulate RNF19A function in resistant cells.

• Pathway reactivation analysis: Develop multiplex detection systems incorporating biotin-conjugated RNF19A antibodies to monitor HR pathway reactivation during acquired resistance.

• Post-translational modification mapping: Use biotin-conjugated antibodies to immunoprecipitate RNF19A from resistant cells and identify altered modification patterns through mass spectrometry.

Understanding how resistance mechanisms affect or bypass RNF19A-mediated regulation of HR could reveal new therapeutic vulnerabilities and inform combination treatment strategies to overcome resistance .

What novel therapeutic strategies might emerge from better understanding RNF19A biology?

Advanced understanding of RNF19A biology could lead to several therapeutic innovations:

• Direct RNF19A modulators: Develop small molecules that enhance RNF19A activity to increase BARD1 ubiquitination, potentially sensitizing HR-proficient cancers to PARP inhibitors.

• Synthetic lethality exploitation: Identify novel synthetic lethal interactions with RNF19A expression levels that could be therapeutically targeted.

• Engineered antibody therapeutics: Create cell-penetrating versions of RNF19A antibodies that could modulate its function intracellularly.

• PROTAC development: Design proteolysis-targeting chimeras that specifically degrade RNF19A in contexts where its activity promotes cancer progression.

• Biomarker-guided therapy selection: Implement RNF19A expression or activity as a biomarker for stratifying patients for DNA damage response-targeted therapies.

• Targeted nanodelivery systems: Develop nanoparticles that recognize cancer cells based on their RNF19A expression pattern using biotin-conjugated antibodies.

Research has shown that RNF19A expression levels affect cancer cell sensitivity to PARP inhibitors, suggesting that pharmacological modulation of RNF19A activity could be a viable approach for enhancing the efficacy of these therapies .