SWI5 Antibody, HRP conjugated

What is SWI5 protein and why is it important in research?

SWI5 is a component of the SWI5-SFR1 complex, which plays a crucial role in double-strand break repair via homologous recombination. This protein is essential for maintaining genomic stability, as it facilitates DNA repair mechanisms that prevent mutations and chromosomal abnormalities. The human SWI5 protein (UniProt ID: Q1ZZU3) is also known as DNA repair protein SWI5 homolog, HBV DNAPTP1-transactivated protein A, or Protein SAE3 homolog. Understanding SWI5 function is particularly important in cancer research, DNA damage response studies, and investigations into genomic integrity maintenance mechanisms .

What are the key specifications of commercially available SWI5 antibodies with HRP conjugation?

Commercially available SWI5 antibodies with HRP conjugation typically target specific amino acid regions of the human SWI5 protein. Most preparations specifically target the amino acid region 54-112 of the human SWI5 protein. These antibodies are commonly polyclonal, derived from rabbit hosts, and purified using Protein G chromatography to achieve >95% purity. They are supplied in liquid format, typically in buffers containing preservatives (such as 0.03% Proclin 300), 50% glycerol, and PBS at pH 7.4. The primary application for these conjugated antibodies is ELISA, with cross-reactivity specifically designed for human samples .

How does HRP conjugation enhance antibody functionality in detection assays?

Horseradish peroxidase (HRP) conjugation transforms antibodies into powerful detection tools by enabling colorimetric signal generation in various immunoassays. The conjugation process creates a direct covalent bond between the HRP enzyme and the antibody, allowing for immediate signal generation upon antigen binding without requiring secondary detection reagents. This direct labeling approach offers several advantages: reduced assay time, elimination of cross-reactivity issues associated with secondary antibodies, lower background signal, and enhanced detection sensitivity. The enzymatic activity of HRP catalyzes the oxidation of chromogenic substrates (such as TMB) in the presence of hydrogen peroxide, producing a colored product that can be quantified spectrophotometrically. This colorimetric reaction provides a reliable and sensitive detection method for antigen-antibody interactions in various research applications .

What are the critical factors affecting the performance of SWI5 antibody, HRP conjugated in ELISA applications?

The performance of SWI5 antibody, HRP conjugated in ELISA applications depends on several critical factors that researchers should carefully control. First, antibody concentration significantly impacts signal-to-noise ratio; optimal concentration ranges between 0.25-1.0 μg/ml based on experimental validation curves. Second, buffer composition affects both antibody stability and enzymatic activity of the HRP moiety - sodium azide must be strictly avoided as it irreversibly inhibits HRP activity. Third, incubation conditions including temperature (optimal at 25°C), time (typically 1-2 hours), and plate coating parameters will influence binding efficiency and specificity. Fourth, proper washing procedures are essential to remove unbound antibody while preserving specific interactions. Fifth, substrate selection and development time must be optimized - TMB substrate with 20-minute development time at room temperature typically yields optimal results before reading absorbance at 450nm. Finally, antibody storage conditions significantly impact long-term performance - storage at -20°C in 50% glycerol with minimal freeze-thaw cycles preserves conjugate activity and specificity .

How should researchers design validation experiments for SWI5 antibody, HRP conjugated?

Researchers should implement a systematic validation approach for SWI5 antibody, HRP conjugated to ensure experimental reliability. Begin with concentration titration experiments using direct ELISA format with immobilized recombinant SWI5 protein (especially covering the 54-112 amino acid region) at concentrations between 0.25-1.0 μg/ml. Generate complete titration curves to determine optimal signal-to-noise ratio and detection limits. Include negative controls using unrelated proteins to assess non-specific binding. Validate specificity using competitive binding assays, where pre-incubation with free SWI5 protein should inhibit antibody binding in a concentration-dependent manner. For cellular applications, include knockout or knockdown cell lines as critical negative controls. When possible, compare results with alternative detection methods such as Western blotting with unconjugated antibodies. Document all validation parameters including incubation times, temperatures, buffer compositions, and substrate development conditions. Finally, prepare a validation report with titration curves, specificity assessments, and reproducibility metrics to guide future experimental designs .

What are the optimal storage conditions for maintaining SWI5 antibody, HRP conjugated activity over time?

Optimal storage conditions for maintaining SWI5 antibody, HRP conjugated activity require careful attention to several parameters to preserve both antibody binding capacity and HRP enzymatic function. The conjugated antibody should be stored at -20°C in aliquots to minimize freeze-thaw cycles, as repeated freezing and thawing can significantly degrade both antibody structure and HRP activity. The storage buffer composition is crucial, typically containing 50% glycerol, 0.01M PBS at pH 7.4, and a preservative such as 0.03% Proclin 300. This formulation provides cryoprotection while maintaining protein stability and preventing microbial growth. Sodium azide must never be used as a preservative with HRP-conjugated antibodies as it irreversibly inhibits HRP activity even at low concentrations. For working solutions, store at 4°C for no more than 2 weeks. Monitoring activity over time is recommended using control ELISA experiments with consistent antigen concentrations to track potential degradation. When storing for extended periods (>6 months), validation experiments should be performed to confirm retained activity before use in critical experiments .

How can researchers determine if their SWI5 antibody, HRP conjugation was successful?

Researchers can employ multiple complementary approaches to verify successful SWI5 antibody, HRP conjugation. The most direct method involves using a quick immunochromatography test, such as an HRP Antibody Conjugation Check Kit, which confirms conjugation without specialized equipment in approximately 30 seconds. Additionally, researchers should perform spectrophotometric analysis measuring the absorbance ratio at 280nm (protein) versus 403nm (HRP) to determine the degree of conjugation. A successful conjugate will show characteristic absorption peaks at both wavelengths. Functional verification through direct ELISA using serial dilutions (1:100 to 1:10,000) of the conjugate against immobilized SWI5 antigen provides a practical assessment of activity. SDS-PAGE analysis under non-reducing conditions can visually confirm conjugation by demonstrating a molecular weight shift compared to unconjugated antibody. For more comprehensive characterization, size exclusion chromatography can separate conjugated antibodies from free antibody or free HRP, revealing conjugation efficiency. Each verification method provides complementary information, and researchers should employ at least two different approaches for confident validation of their conjugation process .

What are the common causes of false positive and false negative results when using SWI5 antibody, HRP conjugated, and how can they be mitigated?

Common causes of false positives include:

• Cross-reactivity with structurally similar proteins, particularly within the DNA repair protein family

• Non-specific binding to plastic surfaces or blocking reagents

• Insufficient washing steps allowing retention of unbound antibody

• Excessive antibody concentration saturating the detection system

• Contamination of reagents with exogenous HRP or peroxidase-like activity

Mitigation strategies for false positives:

• Implement stringent blocking with 5% BSA or casein-based blockers rather than serum

• Extend washing steps using PBS-T (0.05% Tween-20) with at least 5 washes per cycle

• Titrate antibody concentration carefully (optimal range 0.25-1.0 μg/ml)

• Include antigen-free control wells in ELISA plates

• Add HRP inhibitors like sodium azide to wash buffers (but not to antibody storage buffer)

Common causes of false negatives include:

• Degradation of HRP activity due to improper storage or presence of inhibitors

• Insufficient antigen concentration or accessibility

• Competing binding interactions blocking epitope recognition

• Inadequate substrate incubation time or expired substrate

• Improper pH conditions affecting either antibody binding or HRP activity

Mitigation strategies for false negatives:

• Verify HRP activity using direct enzyme activity assays

• Optimize antigen coating concentration and buffer conditions

• Test multiple antibody concentrations in parallel

• Extend substrate development time under controlled conditions

• Maintain pH within optimal range (typically 7.2-7.6) for both antibody binding and HRP activity

Implementation of these targeted troubleshooting approaches can significantly improve the reliability and reproducibility of experiments using SWI5 antibody, HRP conjugated .

What quality control metrics should be established for batch-to-batch consistency of SWI5 antibody, HRP conjugated preparations?

Establishing robust quality control metrics for batch-to-batch consistency of SWI5 antibody, HRP conjugated preparations is essential for reliable research outcomes. A comprehensive QC protocol should include the following parameters:

Each new batch should be compared against a well-characterized reference standard using standardized protocols. Certificate of Analysis documentation should include numerical values for each parameter with defined acceptance ranges. Implementing these quality control metrics ensures consistent performance across experiments and enhances data reproducibility in research settings .

How does the SWI5-SFR1 complex function in DNA repair mechanisms, and how can SWI5 antibody, HRP conjugated contribute to research in this field?

The SWI5-SFR1 complex serves as a critical mediator in homologous recombination (HR) during double-strand break (DSB) repair, a fundamental mechanism protecting genomic integrity. Mechanistically, the complex functions as a co-factor that enhances RAD51 recombinase activity, promoting strand invasion during the HR process. SWI5 specifically interacts with SFR1 to form a heterodimeric complex that stabilizes RAD51 nucleoprotein filaments on single-stranded DNA, facilitating homology search and strand exchange events. This process is crucial for error-free repair of potentially lethal DSBs that might otherwise lead to chromosomal rearrangements or cell death.

SWI5 antibody, HRP conjugated provides valuable tools for investigating this complex through multiple experimental approaches. In chromatin immunoprecipitation (ChIP) assays, the conjugated antibody can directly detect SWI5 recruitment to DSB sites without requiring secondary antibody steps, increasing assay efficiency and reducing background. For immunofluorescence microscopy, the HRP conjugate enables sensitive detection of SWI5 localization during different cell cycle phases and in response to DNA-damaging agents through tyramide signal amplification protocols. In protein interaction studies, the conjugated antibody facilitates pull-down experiments to identify novel SWI5 binding partners within the DNA repair machinery. Importantly, quantitative ELISAs using the HRP-conjugated antibody can measure changes in SWI5 expression levels across different cell types, cancer models, or following exposure to genotoxic agents.

Researchers in the DNA repair field can leverage these tools to address fundamental questions: How is SWI5-SFR1 recruitment regulated in response to different types of DNA damage? What post-translational modifications modulate SWI5 function during the repair process? How do cancer-associated mutations impact SWI5-SFR1 complex formation and function? Answers to these questions could reveal new therapeutic vulnerabilities in cancers with defective HR repair pathways .

What are the advanced considerations for using SWI5 antibody, HRP conjugated in multiplex detection systems?

Implementing SWI5 antibody, HRP conjugated in multiplex detection systems requires addressing several sophisticated technical considerations to ensure reliable and interpretable results. First, researchers must carefully evaluate substrate compatibility when combining HRP-based detection with other enzymatic or fluorescent reporters. For systems using multiple HRP-conjugated antibodies, differential substrate development is essential - consider sequential application of spectrally distinct substrates with interim signal acquisition, or utilize tyramide signal amplification with distinct fluorophores to spatially resolve signals. Second, potential cross-reactivity must be systematically assessed, not only against target antigens but also between detection reagents themselves; perform extensive validation using single-analyte controls alongside multiplexed samples to identify potential interference patterns. Third, dynamic range optimization becomes critical in multiplex systems - establish individual titration curves for each conjugated antibody to determine concentration ranges where signals remain proportional to analyte concentration without saturating detection systems. Fourth, carefully evaluate temporal dynamics of enzymatic reactions, as substrate depletion from one highly abundant target may affect detection sensitivity for other targets. Fifth, incorporate internal normalization controls specifically designed for the multiplex format to account for well-to-well or sample-to-sample variation. Finally, develop custom analysis algorithms that can deconvolute overlapping signals and correct for spectral overlap when multiplexing with other fluorescent or colorimetric reporters. These advanced considerations will maximize the information gained from complex biological samples while maintaining quantitative accuracy across multiple analytes .

What is the recommended protocol for preparing SWI5 antibody, HRP conjugated in a research laboratory?

The following protocol outlines a research-grade method for preparing SWI5 antibody, HRP conjugated using modern bioconjugation technology that preserves both antibody specificity and HRP activity:

Materials Required:

• Purified SWI5 antibody (1-4 mg/ml in amine-free buffer)

• Activated 4FB-HRP (pre-activated horseradish peroxidase)

• 10-50 mM amine-free buffer (HEPES, MES, or phosphate buffer, pH 6.5-8.5)

• TurboLink Catalyst Buffer

• Spin columns for purification

• UV-Vis spectrophotometer for conjugate analysis

Procedure:

• Antibody Preparation: Ensure SWI5 antibody is in compatible buffer (10-50 mM amine-free buffer, pH 6.5-8.5) at concentration 1-4 mg/ml. Avoid buffers with primary amines, thiols, or sodium azide.

• Determine optimal HRP:Antibody ratio: For most applications, use molar ratios between 1:4 and 1:1 (antibody:HRP). Accounting for molecular weights (160,000 vs 40,000), this translates to using 1-4 mg antibody per 1 mg HRP.

• Modification of Antibody:
  a. Add HyNic modification reagent to antibody solution at 10-15 molar excess
  b. Incubate for 2 hours at room temperature
  c. Remove excess reagents using provided spin columns

• Conjugation Reaction:
  a. Combine HyNic-modified antibody with 4FB-activated HRP
  b. Add TurboLink Catalyst Buffer to reaction (10% of total volume)
  c. Incubate for 2-4 hours at room temperature

• Purification:
  a. Apply reaction mixture to size exclusion spin column
  b. Centrifuge according to column specifications
  c. Collect purified conjugate

• Analysis and Characterization:
  a. Determine protein concentration using absorbance at 280 nm
  b. Measure HRP incorporation using absorbance at 403 nm
  c. Calculate conjugation ratio using molar extinction coefficients

• Storage:
  a. Add glycerol to 50% final concentration
  b. Add preservative (0.03% Proclin 300, not sodium azide)
  c. Store in aliquots at -20°C

This protocol leverages SoluLINK bioconjugation technology which forms stable bonds without compromising antibody or enzyme function, resulting in high-activity conjugates suitable for sensitive detection applications. The TurboLink catalyst significantly accelerates reaction kinetics, ensuring quantitative conversion under mild conditions .

What optimization strategies can improve signal-to-noise ratio when using SWI5 antibody, HRP conjugated in immunoassays?

Optimizing signal-to-noise ratio when using SWI5 antibody, HRP conjugated requires systematic refinement of multiple experimental parameters. First, buffer optimization is critical - incorporate 0.1-0.5% non-ionic detergent (such as Tween-20) in wash and diluent buffers to reduce non-specific binding, while maintaining ionic strength between 150-300 mM to preserve specific interactions. Second, implement strategic blocking approaches using 5% BSA or commercially optimized blocking reagents with 1-2 hour incubation at room temperature to saturate non-specific binding sites. Third, titrate antibody concentration precisely using 2-fold serial dilutions from 2 μg/ml to 0.125 μg/ml against constant antigen concentration to identify optimal concentration that maximizes specific signal while minimizing background. Fourth, extend and optimize washing protocols to include at least 5 washes of 5 minutes each with gentle agitation to thoroughly remove unbound conjugate. Fifth, implement signal development optimization by testing multiple substrates (TMB, ABTS, or enhanced chemiluminescent reagents) with controlled temperature and timed development to maximize detection sensitivity. Sixth, consider signal amplification methods such as avidin-biotin systems or tyramide signal amplification compatible with HRP detection when working with low-abundance targets. Finally, incorporate rigorous controls including antigen-negative wells, isotype-matched irrelevant antibody-HRP conjugates, and substrate-only controls to clearly distinguish specific signal from background noise. These methodical approaches should be sequentially implemented, with careful documentation of improvement at each step, to achieve optimal signal-to-noise performance across different experimental contexts .

How can researchers validate SWI5 antibody, HRP conjugated specificity in complex biological samples?

Validating SWI5 antibody, HRP conjugated specificity in complex biological samples requires a multi-faceted approach that confirms target recognition while ruling out cross-reactivity. Begin with epitope competition assays, where pre-incubation of the conjugated antibody with excess recombinant SWI5 protein (specifically the 54-112 amino acid region) should abolish specific signals in subsequent detection assays. This blocking effect establishes epitope-specific binding. Proceed with genetic validation using SWI5 knockdown or knockout models - testing the conjugated antibody in wild-type versus SWI5-depleted samples should demonstrate signal reduction proportional to target reduction. For tissue samples, perform parallel staining with an alternative SWI5 antibody raised against a different epitope; concordant staining patterns substantiate target specificity.

Implement orthogonal validation by comparing protein detection across multiple platforms - correlate ELISA results with Western blot, immunohistochemistry, or mass spectrometry data to confirm consistent target identification. For enrichment approaches, perform pull-down experiments followed by mass spectrometry to identify all proteins captured by the antibody, ensuring SWI5 ranks as the predominant hit. Evaluate cross-reactivity systematically by testing against recombinant proteins with sequence homology to SWI5, particularly other DNA repair proteins, establishing specificity boundaries.

Finally, perform immunoprecipitation followed by Western blotting with a second SWI5 antibody targeting a different epitope to confirm the identity of captured proteins. Document all validation experiments comprehensively, including methodology, controls, and quantitative assessments of specificity metrics to establish confidence in the conjugated antibody's performance across experimental systems .