INTS3 Antibody, HRP conjugated

What is INTS3 and why is it a significant research target?

INTS3 (Integrator Complex Subunit 3) is a critical component of the SOSS1 complex involved in DNA damage response pathways. Its significance stems from its interaction with single-stranded DNA-binding protein 1 (hSSB1), which plays a crucial role in maintaining genomic stability through DNA repair mechanisms. Researchers target INTS3 to understand its function in DNA damage response and to potentially develop therapeutic approaches for conditions characterized by genomic instability, such as cancer. The protein-protein interaction (PPI) between INTS3 and hSSB1 has been identified as a potential therapeutic target, particularly in the context of DNA damage repair mechanisms .

What are the key applications for INTS3 antibodies in research settings?

INTS3 antibodies are valuable tools for investigating protein expression, localization, and interactions in various experimental contexts. The HRP-conjugated variants specifically enable enhanced sensitivity in detection assays. Common applications include ELISA for quantitative protein detection, western blotting for protein expression analysis, immunofluorescence for cellular localization studies, and immunoprecipitation for protein-protein interaction investigations. When studying the INTS3-hSSB1 complex, these antibodies can be utilized in co-immunoprecipitation assays to evaluate protein-protein interactions under various experimental conditions, such as after treatment with potential inhibitory compounds .

How should I select between different epitope-specific INTS3 antibodies?

Selection of epitope-specific antibodies depends on your experimental objectives and the protein domain of interest. For INTS3, various antibodies target different amino acid regions, such as AA 901-1043, AA 518-551, AA 960-998, and AA 1000-1034. When studying INTS3-hSSB1 interaction, consider antibodies targeting the N-terminal region of INTS3, as this contains important binding interfaces with hSSB1. The AA 901-1043 epitope region covered by the HRP-conjugated antibody in the catalog provides recognition of the C-terminal region, which may have different functional significance than the N-terminal region involved in protein-protein interactions. Review literature on the functional domains of INTS3 to determine which epitope best aligns with your specific research question .

What is the significance of HRP conjugation in INTS3 antibodies?

Horseradish peroxidase (HRP) conjugation provides significant advantages for detection sensitivity and versatility in various assays. The enzyme catalyzes colorimetric, chemiluminescent, or fluorescent reactions depending on the substrate used, enabling flexible detection strategies. In ELISA applications, HRP-conjugated INTS3 antibodies offer enhanced sensitivity through signal amplification, allowing detection of low abundance proteins. The conjugation eliminates the need for secondary antibodies in many applications, streamlining experimental workflows and reducing background signal. When designing experiments, consider that HRP-conjugated antibodies are optimized for detection methods rather than functional assays that might require unconjugated antibodies .

How can I optimize immunoprecipitation protocols when studying INTS3-hSSB1 interactions?

Optimizing immunoprecipitation for INTS3-hSSB1 interaction studies requires careful consideration of buffer conditions, antibody specificity, and experimental controls. Begin with cell lysis using a buffer containing 150 mM NaCl, with mild detergents like 0.1-0.5% NP-40 or Triton X-100 to preserve protein-protein interactions. Pre-clear lysates with protein G beads to reduce non-specific binding. For INTS3-hSSB1 co-immunoprecipitation, the published methods indicate success using U2OS cell lines and hSSB1 antibodies for the pull-down, as demonstrated in studies investigating compounds that disrupt this interaction. When evaluating compounds that may disrupt INTS3-hSSB1 interaction, treat cells for approximately 36 hours prior to lysis and immunoprecipitation. Include appropriate controls: IgG control for antibody specificity, input control (typically 5% of lysate) for expression levels, and untreated/vehicle controls when testing compounds .

What methodological approaches can detect subtle changes in INTS3-hSSB1 interactions under different experimental conditions?

Detecting subtle changes in INTS3-hSSB1 interactions requires sensitive and quantitative techniques beyond standard co-immunoprecipitation. Quantitative co-immunoprecipitation followed by densitometric analysis of western blots can provide semi-quantitative assessment of interaction strength. This method was successfully employed to evaluate how compounds I1-I5 affected INTS3-hSSB1 association, revealing that compounds I3, I4, and I5 reduced this interaction. For higher sensitivity, consider employing biophysical techniques such as surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) with recombinant proteins to directly measure binding affinities and kinetics. These methods would provide quantitative data on how experimental conditions or test compounds affect binding parameters. Proximity ligation assays offer another option for visualizing protein interactions within intact cells with high sensitivity, providing spatial information about where interactions occur under different experimental conditions .

How can molecular dynamics simulations complement experimental data when studying INTS3 protein interactions?

Molecular dynamics (MD) simulations provide valuable structural insights that complement experimental techniques when investigating INTS3 protein interactions. For INTS3-hSSB1 interactions, MD simulations can reveal binding stability, conformational changes, and energetic contributions of specific amino acid residues. Research has demonstrated that MD simulations run for 20-25 ns at NPT ensembles (constant number, pressure, and temperature) effectively evaluate protein-ligand stability. These simulations should include analysis of root mean square deviation (RMSD) to assess structural stability and root mean square fluctuations (RMSF) to identify regions of conformational flexibility. Binding free energy calculations using methods like MM-GBSA provide quantitative measures of interaction strength, correlating with experimental findings. The specific implementation includes system equilibration under restraints (gradually reduced from 5.0 to 0.1 kcal/(mol Ų)), and using particle mesh Ewald methods for long-range electrostatic interactions .

What strategies can be employed to validate INTS3-targeting compounds identified through virtual screening?

Validating INTS3-targeting compounds identified through virtual screening requires a multi-tiered approach combining computational refinement and experimental validation. Begin with rescoring high-ranking compounds using MM-GBSA calculations following 20 ns molecular dynamics simulations to improve prediction accuracy for binding affinity. The research shows this approach successfully identified compounds I4 and I5 as having the strongest predicted binding to INTS3. For experimental validation, employ co-immunoprecipitation assays in appropriate cell lines (such as U2OS cells) to assess disruption of protein-protein interactions, as demonstrated in the research where compounds I3, I4, and I5 reduced INTS3-hSSB1 association. Conduct cell-based immunofluorescence staining to visualize effects on protein localization, particularly at DNA damage sites. For definitive binding confirmation, perform biophysical assays with recombinant proteins, such as SPR or ITC, to establish direct binding and determine affinity constants. These orthogonal approaches provide comprehensive validation by connecting computational predictions with cellular effects .

What controls are essential when performing western blotting with HRP-conjugated INTS3 antibodies?

When performing western blotting with HRP-conjugated INTS3 antibodies, several essential controls must be implemented to ensure reliable and interpretable results. Include a positive control sample with known INTS3 expression, such as recombinant INTS3 protein or lysate from a cell line with validated expression. Run a negative control sample from cells with INTS3 knockdown or from tissues/cells known not to express the protein. Incorporate loading controls using housekeeping proteins (e.g., β-actin, GAPDH) to normalize for sample loading variations. To control for antibody specificity, pre-block the antibody with recombinant INTS3 peptide (immunogen competition) prior to membrane incubation. When evaluating experimental treatments affecting INTS3, include appropriate vehicle control treatments. For HRP-conjugated antibodies specifically, include an enzyme activity control by testing substrate reaction without primary antibody to assess background signal. These controls collectively ensure the validity of results obtained with the HRP-conjugated INTS3 antibody .

How can researchers accurately assess INTS3 localization in response to DNA damage?

Accurately assessing INTS3 localization in response to DNA damage requires careful experimental design and appropriate imaging techniques. Employ immunofluorescence using appropriate fixation methods (typically 4% paraformaldehyde followed by permeabilization) with the INTS3 antibody. When using HRP-conjugated antibodies, convert to fluorescence detection using tyramide signal amplification. Induce DNA damage using standardized methods such as ionizing radiation (2-10 Gy), UV irradiation, or chemical agents like etoposide or hydroxyurea. Include time-course analysis post-damage induction (typically 15 minutes to 24 hours) to capture dynamic relocalization events. Co-stain for established DNA damage markers such as γH2AX to confirm damage sites and assess colocalization with INTS3. Use confocal microscopy with z-stack imaging to accurately determine three-dimensional localization patterns. For quantitative assessment, measure parameters such as foci number, intensity, and colocalization coefficients with DNA damage markers across multiple cells (n ≥ 50) and experimental replicates. This methodological approach enables reliable assessment of how INTS3 localization changes in response to genomic stress .

What methodological approaches best determine if INTS3-targeting compounds affect DNA damage responses?

To determine if INTS3-targeting compounds affect DNA damage responses, researchers should implement a multi-faceted methodological approach. Begin with comet assays to quantify DNA damage levels in cells treated with candidate compounds, comparing results to appropriate controls. Measure DNA damage signaling by western blotting for phosphorylated markers (γH2AX, phospho-ATM, phospho-CHK2) at various time points after damage induction. Assess DNA repair kinetics through immunofluorescence time-course experiments tracking resolution of γH2AX foci following damage induction in the presence or absence of compounds. Evaluate functional consequences using clonogenic survival assays following DNA damage, comparing compound-treated versus control cells. For mechanistic insights, perform chromatin immunoprecipitation to determine if compounds affect recruitment of repair factors to damage sites. Cell cycle analysis by flow cytometry can reveal whether compounds affect damage-induced checkpoint activation. The research suggests that compounds disrupting INTS3-hSSB1 interaction prevented recruitment of these proteins to DNA damage sites, indicating that these methodological approaches can effectively evaluate compound effects on DNA damage response pathways .

How should researchers address non-specific binding when using HRP-conjugated INTS3 antibodies?

Non-specific binding with HRP-conjugated INTS3 antibodies requires systematic troubleshooting and optimization strategies. Begin by increasing blocking stringency using 5% BSA or milk in TBS-T, with longer blocking times (2-3 hours at room temperature or overnight at 4°C). Optimize antibody dilution through titration experiments, typically testing a range from 1:500 to 1:5000, as appropriate dilutions balance specific signal with background reduction. For western blotting applications, increase washing duration and frequency (5-6 washes of 10 minutes each) using TBS-T with higher detergent concentration (0.1-0.2% Tween-20). When persistent non-specific bands occur, add reducing agents such as 5 mM DTT to sample buffer and consider protein A/G pre-clearing of lysates. For immunohistochemistry or immunofluorescence, include 0.1-0.3% Triton X-100 in antibody diluent to reduce non-specific membrane binding. The high purity (>95%) of the Protein G-purified HRP-conjugated INTS3 antibody should minimize non-specific binding, but these optimization steps may still be necessary for challenging sample types or applications .

What are effective strategies for enhancing signal detection when INTS3 expression is low?

When INTS3 expression is low, researchers can employ several strategies to enhance signal detection with HRP-conjugated antibodies. Increase protein loading amounts (up to 50-100 μg per lane for western blotting) while maintaining appropriate running and transfer conditions. Utilize enhanced chemiluminescence (ECL) substrates specifically designed for high sensitivity, such as femto-level ECL reagents that can improve detection limits by 10-100 fold. For western blotting, consider extended exposure times with high-sensitivity imaging systems or X-ray film. Implement signal amplification methods such as tyramide signal amplification (TSA) to enhance HRP signal output, which can increase sensitivity by up to 100-fold in immunohistochemistry and immunofluorescence applications. When working with cells, consider pre-treatment with proteasome inhibitors (e.g., MG132) for 4-6 hours before harvest to prevent protein degradation and increase detectable levels. For ELISA applications, extend substrate incubation times and optimize plate-coating conditions. These methods collectively enhance detection sensitivity while maintaining signal specificity when working with samples containing low INTS3 expression levels .

How can researchers accurately quantify changes in INTS3 expression or localization across experimental conditions?

Accurate quantification of changes in INTS3 expression or localization requires rigorous analytical approaches and appropriate normalization methods. For western blot analysis, perform densitometry using specialized software (ImageJ, Image Lab) with background subtraction, normalizing INTS3 band intensity to loading controls (GAPDH, β-actin). Always operate within the linear range of detection, establishing this range through standard curve analysis with recombinant protein or serial dilutions of positive control samples. For immunofluorescence quantification, measure parameters including mean fluorescence intensity, nuclear/cytoplasmic ratio, and foci number per cell across multiple fields (minimum 10) and experimental replicates (minimum 3). Employ automated image analysis workflows with consistent thresholding parameters to reduce subjective bias. When assessing treatment effects, calculate fold change relative to control conditions rather than absolute values, accounting for inter-experimental variability. For complex localization patterns, consider advanced analysis methods such as Pearson's correlation coefficient for co-localization studies or fractal dimension analysis for pattern distribution. Statistical analysis should include appropriate tests (t-test, ANOVA) with multiple testing correction when comparing across numerous conditions .

What bioinformatic approaches can help interpret INTS3 interaction data in the context of DNA damage response pathways?

Interpreting INTS3 interaction data within DNA damage response pathways benefits from integrative bioinformatic approaches that contextualize experimental findings. Employ protein-protein interaction network analysis using databases like STRING, BioGRID, or IntAct to place INTS3-hSSB1 interactions within broader DNA repair networks. The research demonstrates that INTS3-hSSB1 interaction is crucial for the SOSS1 complex function in DNA damage response. Perform pathway enrichment analysis using tools like KEGG, Reactome, or Gene Ontology to identify biological processes associated with INTS3 interacting partners. For structural insights, utilize molecular docking and dynamics simulations to predict how mutations or compounds might affect interaction interfaces, as demonstrated in the research where compounds targeting specific grooves between α-helices of INTS3's N-terminus disrupted hSSB1 binding. When analyzing the effects of experimental perturbations (e.g., compounds I3-I5), use differential network analysis to identify altered interaction patterns. Integration of experimental interaction data with publicly available multi-omics datasets through platforms like cBioPortal can reveal correlations between INTS3 interactions and clinical outcomes in diseases like cancer, providing broader biological context for mechanistic studies .