SWI5 Antibody, Biotin conjugated

What is SWI5 protein and what cellular functions does it serve?

SWI5 (also known as C9orf119 or SAE3 homolog) is a DNA repair protein that functions as a critical component of the SWI5-SFR1 complex. This complex plays an essential role in the repair of double-strand breaks (DSBs) via homologous recombination, a vital cellular process that maintains genomic integrity . The protein is encoded by the SWI5 gene (GeneID: 375757) and has been assigned the UniProt primary accession number Q1ZZU3 . Through its participation in homologous recombination, SWI5 helps cells accurately repair DNA damage that could otherwise lead to mutations, chromosomal aberrations, or cell death. Understanding this protein's function is crucial for researchers investigating DNA damage response pathways, cancer biology, and genomic stability mechanisms.

What is the significance of biotin conjugation in SWI5 antibodies?

Biotin conjugation represents a strategic modification that significantly enhances the utility of SWI5 antibodies in research applications. This conjugation process chemically links biotin molecules to the antibody structure, creating a powerful detection tool that leverages the exceptionally strong biotin-streptavidin interaction (one of the strongest non-covalent biological interactions known) . The biotin tag serves as an intermediary binding site that can be recognized by streptavidin conjugated to various reporter molecules such as enzymes, fluorophores, or nanoparticles. This arrangement provides multiple advantages including signal amplification (as multiple streptavidin molecules can bind to a single biotinylated antibody), flexibility in detection strategies, and enhanced sensitivity compared to directly labeled antibodies. For SWI5 detection, which may be expressed at relatively low levels in some cellular contexts, this sensitivity enhancement is particularly valuable for accurate quantification and localization studies.

What are the optimal experimental conditions for using biotin-conjugated SWI5 antibody in ELISA?

Optimizing ELISA protocols with biotin-conjugated SWI5 antibody requires careful attention to several experimental parameters. Based on product specifications, the recommended dilution range for ELISA applications is typically 1:500-1:1000, though researchers should perform titration experiments to determine the optimal concentration for their specific application . The protocol should include proper blocking steps (typically using 1-5% BSA or non-fat dry milk) to minimize background signal. For detection, streptavidin-HRP (horseradish peroxidase) should be diluted according to manufacturer recommendations (typically 1:1000 to 1:5000) and incubated for 30-60 minutes at room temperature. Temperature control is critical throughout the procedure, with antigen coating and primary antibody incubation typically performed at 4°C overnight for maximum sensitivity, while detection steps are usually carried out at room temperature. Washing steps should be thorough (3-5 washes with PBS-T) to remove unbound antibodies. When developing the signal with TMB substrate, monitor the reaction closely and stop it with sulfuric acid solution when appropriate colorimetric development is observed, typically within 5-15 minutes. This methodical approach ensures maximum sensitivity while maintaining low background signals.

How can I design a dual-labeling experiment incorporating SWI5 biotin-conjugated antibody?

Designing effective dual-labeling experiments with biotin-conjugated SWI5 antibody requires careful selection of compatible secondary detection systems and experimental controls. Begin by selecting a complementary antibody target that localizes to a distinct cellular compartment or marks a different cell population than SWI5. This second antibody should be from a different host species than rabbit to avoid cross-reactivity. For example, a mouse monoclonal antibody against a nuclear marker could complement the SWI5 detection. For detection, employ a streptavidin conjugate with one fluorophore (e.g., streptavidin-Cy3) for SWI5 visualization, while using a species-specific secondary antibody with a spectrally distinct fluorophore (e.g., anti-mouse IgG-Alexa Fluor 488) for the second target. Critical controls should include: (1) single-label controls to verify no spectral bleed-through between channels, (2) secondary-only controls to assess non-specific binding, and (3) pre-absorption controls where the SWI5 antibody is pre-incubated with recombinant SWI5 protein to confirm specificity. The staining protocol should be sequential rather than simultaneous to prevent potential interference between detection systems, with careful optimization of incubation times and concentrations for each reagent.

What methodologies can enhance the detection sensitivity when working with biotin-conjugated SWI5 antibody?

Several methodological approaches can significantly enhance detection sensitivity when working with biotin-conjugated SWI5 antibody. First, consider implementing a tyramide signal amplification (TSA) system, which utilizes streptavidin-HRP followed by catalyzed deposition of biotinylated tyramide, potentially increasing sensitivity by 10-100 fold over conventional detection methods. Second, employ a multi-layered detection approach by using streptavidin-biotin complexes (ABC method) where alternating layers of streptavidin and biotinylated detection molecules create signal amplification. Third, optimize antigen retrieval protocols if working with fixed tissues or cells – for formalin-fixed samples, try citrate buffer (pH 6.0) heat-induced epitope retrieval at 95-100°C for 20 minutes followed by 20 minutes cooling. Fourth, extend primary antibody incubation times to 48-72 hours at 4°C with gentle agitation to improve antibody penetration and binding in thick tissue sections. Fifth, use carrier proteins (0.1-0.5% BSA) in dilution buffers to prevent non-specific antibody adherence to tubes and plates. Finally, consider sample pre-clearing with protein A/G beads to reduce background caused by endogenous immunoglobulins. These methodological refinements can collectively enhance detection sensitivity by several orders of magnitude compared to standard protocols.

How can I address high background issues when using biotin-conjugated SWI5 antibody?

High background signal when using biotin-conjugated SWI5 antibody often stems from several identifiable sources that can be systematically addressed through methodological adjustments. First, endogenous biotin in biological samples can be a major contributor to non-specific signals. Implement a biotin blocking step using commercially available biotin blocking kits (typically employing avidin followed by biotin) before applying the primary antibody. Second, insufficient blocking can lead to non-specific antibody binding – increase blocking agent concentration to 3-5% and extend blocking time to 2 hours at room temperature. Third, overly concentrated antibody application increases non-specific binding; perform a careful titration series starting from 1:500 dilution and increasing to 1:5000 to identify the optimal signal-to-noise ratio . Fourth, incomplete washing between steps allows residual reagents to contribute to background; increase wash steps to 5 times with 5 minutes of gentle agitation per wash. Fifth, endogenous peroxidase or phosphatase activity can generate signal if using enzyme-based detection systems; pretreat samples with 0.3% H₂O₂ in methanol for 30 minutes to quench endogenous peroxidase activity. Finally, storage conditions of the antibody may lead to aggregation; ensure proper aliquoting and storage at -20°C while avoiding repeated freeze-thaw cycles as recommended in the product specifications .

What are the critical factors to consider when validating SWI5 antibody specificity in different experimental contexts?

Validating SWI5 antibody specificity across different experimental contexts requires a multi-faceted approach that addresses several critical factors. First, implement genetic controls by comparing staining patterns between wild-type samples and SWI5 knockout/knockdown samples – the specific signal should be significantly reduced or absent in the latter. Second, perform peptide competition assays by pre-incubating the antibody with excess recombinant SWI5 protein (54-112AA immunogen region) , which should substantially reduce genuine target recognition. Third, validate across multiple applications – if the antibody shows consistent SWI5 detection patterns in orthogonal techniques (e.g., Western blot, immunoprecipitation, immunofluorescence), specificity is more strongly supported. Fourth, confirm molecular weight correspondence – the detected protein should match the expected molecular weight of SWI5. Fifth, verify subcellular localization patterns against published literature – SWI5 should show appropriate distribution related to its role in DNA repair. Sixth, cross-reference with independent antibodies targeting different epitopes of SWI5 – concordant results strengthen confidence in specificity. Finally, perform mass spectrometry analysis on immunoprecipitated material to confirm the presence of SWI5 and assess any potential cross-reactivities. This comprehensive validation strategy ensures reliable interpretation of experimental results across different contexts.

How does the specific immunogen region (AA 54-112) impact epitope recognition and experimental design?

The specific immunogen region (amino acids 54-112) of the SWI5 protein used to generate this antibody has significant implications for epitope recognition and experimental design . This relatively narrow 59-amino acid region represents only a portion of the full-length human SWI5 protein, suggesting the antibody recognizes a specific domain rather than multiple regions throughout the protein. This specificity has several experimental considerations. First, protein denaturation conditions in applications like Western blotting might expose or conceal this epitope region depending on the specific buffers and detergents used – researchers should optimize denaturation conditions to ensure epitope accessibility. Second, in applications involving protein-protein interactions, binding partners might mask this specific region, potentially resulting in false negatives; epitope accessibility testing with and without interaction partners is advisable. Third, post-translational modifications occurring within the 54-112 region could potentially alter antibody recognition; researchers should determine if known modifications occur in this region and how they might affect detection. Fourth, when designing fusion proteins or tagged constructs, placing tags near this region might interfere with antibody binding. Fifth, for experiments involving protein fragments or truncated variants, this antibody will only detect variants containing the 54-112 region. Understanding these implications allows researchers to design experiments that accurately account for the specific epitope recognition properties of this antibody.

How can I quantitatively analyze SWI5 expression levels using biotin-conjugated antibody in ELISA?

Quantitative analysis of SWI5 expression using biotin-conjugated antibody in ELISA requires rigorous methodological approaches to ensure accurate and reproducible results. First, establish a standard curve using recombinant SWI5 protein at concentrations ranging from 0.1 ng/mL to 1000 ng/mL, prepared through serial dilutions in the same buffer as your samples. Plot the optical density values against known concentrations using a four-parameter logistic curve fit, which accommodates the non-linear relationship typically observed in ELISA. Second, prepare samples at multiple dilutions (typically three dilutions in duplicate) to ensure measurements fall within the linear range of the standard curve. Third, include appropriate controls: negative controls (buffer only), positive controls (samples with known SWI5 expression), and spike-in controls (samples with added recombinant SWI5 at known concentrations) to assess recovery and matrix effects. Fourth, normalize SWI5 measurements to total protein concentration determined by Bradford or BCA assay to account for variation in sample preparation. Fifth, assess intra-assay variability (coefficient of variation between replicates should be <10%) and inter-assay variability (CV between experiments should be <15%). Finally, perform statistical analysis appropriate for your experimental design, such as t-tests for two-group comparisons or ANOVA for multiple groups, with post-hoc corrections for multiple comparisons. This comprehensive approach ensures reliable quantification of SWI5 expression levels.

What approaches can resolve contradictory results between SWI5 protein detection and gene expression data?

Resolving contradictory results between SWI5 protein detection using the biotin-conjugated antibody and gene expression data requires systematic investigation of several potential explanations. First, examine post-transcriptional regulation mechanisms – discrepancies might be explained by microRNA-mediated repression, RNA binding proteins affecting translation efficiency, or alterations in mRNA stability. Quantify SWI5 mRNA stability through actinomycin D chase experiments and assess polysome association to determine translation efficiency. Second, investigate post-translational regulation – increased protein turnover despite normal transcription could explain low protein levels with high mRNA expression. Perform cycloheximide chase experiments to measure SWI5 protein half-life and use proteasome inhibitors (e.g., MG132) to assess degradation pathways. Third, consider epitope accessibility issues – conformational changes, protein complex formation, or post-translational modifications might mask the 54-112AA region recognized by the antibody without affecting mRNA levels. Use alternative antibodies targeting different SWI5 epitopes to confirm this possibility. Fourth, evaluate technical considerations including sample preparation differences between protein and RNA analyses, cell/tissue heterogeneity where bulk RNA measurements might not reflect protein expression in specific cell subpopulations, or temporal differences in collection timing that fail to account for expression dynamics. Finally, validate findings with orthogonal methods such as mass spectrometry for protein quantification and digital droplet PCR for more accurate gene expression measurement. This structured approach helps reconcile seemingly contradictory results between different analytical platforms.

How can SWI5 antibody be utilized to investigate protein-protein interactions within the SWI5-SFR1 complex?

Investigating protein-protein interactions within the SWI5-SFR1 complex using biotin-conjugated SWI5 antibody can be approached through several advanced methodologies. First, implement proximity ligation assay (PLA) by combining the biotin-conjugated SWI5 antibody with an antibody against SFR1 or other suspected interaction partners. This technique generates fluorescent spots only when proteins are within 40nm of each other, providing in situ visualization of interactions with subcellular resolution. Second, perform co-immunoprecipitation studies using the SWI5 antibody coupled to streptavidin beads, followed by mass spectrometry analysis to identify novel binding partners beyond the known SFR1 interaction . Third, develop a protein interaction map through BioID proximity labeling by creating a SWI5-BirA fusion protein that biotinylates proximal proteins, which can then be captured using streptavidin and identified through proteomics. Fourth, assess interaction dynamics following DNA damage by conducting time-course experiments after genotoxic stress induction, combining immunoprecipitation with quantitative proteomics to track temporal changes in the SWI5 interactome. Fifth, determine structural requirements for these interactions by comparing wild-type SWI5 with truncation mutants lacking specific domains, including analysis of the immunogen region (AA 54-112) for its potential role in mediating protein-protein interactions. Finally, validate key interactions through reciprocal co-immunoprecipitation, yeast two-hybrid assays, and functional rescue experiments to establish biological significance. This multi-faceted approach provides comprehensive characterization of SWI5's interaction network within the context of DNA repair mechanisms.

What strategies can integrate SWI5 antibody-based detection with cutting-edge genomic technologies?

Integrating SWI5 antibody-based detection with cutting-edge genomic technologies creates powerful approaches for understanding the functional genomics of DNA repair mechanisms. First, implement CUT&RUN or CUT&Tag protocols using the biotin-conjugated SWI5 antibody to map SWI5 binding sites throughout the genome with higher resolution and lower background than traditional ChIP-seq. The biotin conjugation allows direct capture on streptavidin beads, streamlining the protocol. Second, combine Chromatin Immunoprecipitation (ChIP) using SWI5 antibody with high-throughput sequencing (ChIP-seq) to identify genomic regions where SWI5 binds following DNA damage, potentially revealing preferential binding to specific chromatin contexts or sequence motifs. Third, develop CRISPR activation/inhibition screens targeting genes identified from SWI5 ChIP-seq data to establish functional relationships between genomic binding and biological outcomes. Fourth, implement spatial transcriptomics combined with SWI5 immunofluorescence to correlate SWI5 protein localization with transcriptional changes in the surrounding microenvironment following DNA damage. Fifth, perform nascent RNA-seq after SWI5 antibody-mediated immunoprecipitation of chromatin to identify actively transcribed genes associated with SWI5 binding. Finally, utilize single-cell approaches combining SWI5 antibody-based protein detection with single-cell RNA-seq through CITE-seq technology to correlate SWI5 protein levels with transcriptional profiles at single-cell resolution. These integrated approaches bridge the gap between genomic data and protein function, providing unprecedented insights into SWI5's role in maintaining genomic integrity.

How can mathematical modeling enhance interpretation of SWI5 kinetics in homologous recombination?

Mathematical modeling provides powerful frameworks for interpreting SWI5 kinetics in homologous recombination when integrated with experimental data generated using biotin-conjugated SWI5 antibody. First, develop ordinary differential equation (ODE) models that capture the temporal dynamics of SWI5 recruitment to DNA damage sites, incorporating parameters for association/dissociation rates, complex formation with SFR1, and progression through repair phases. These models can be calibrated using time-course immunofluorescence data quantifying SWI5 localization patterns following DNA damage. Second, implement stochastic modeling approaches (e.g., Gillespie algorithm) to account for the inherent randomness in repair processes when SWI5 is present at low copy numbers in cells. Third, create agent-based models that simulate the spatial organization of repair complexes, using super-resolution microscopy data of biotin-labeled SWI5 to inform spatial parameters and interaction rules. Fourth, develop Bayesian statistical frameworks to integrate heterogeneous data types (protein levels from ELISA, localization data from imaging, interaction data from co-IP) for more robust parameter estimation and uncertainty quantification. Fifth, construct network models connecting SWI5 activity to downstream repair outcomes, allowing simulation of perturbation effects and identification of critical nodes in the repair pathway. These modeling approaches should incorporate sensitivity analysis to identify parameters most influencing system behavior and validation against independent experimental datasets. Through this integration of mathematical modeling with antibody-based experimental approaches, researchers can develop mechanistic insights into SWI5 function that would be difficult to obtain through experiments alone, ultimately guiding the design of targeted interventions in DNA repair pathways.