FLII Antibody, HRP conjugated

What is FLII Antibody, HRP conjugated?

FLII Antibody, HRP conjugated is a high-quality polyclonal antibody that has been chemically linked to horseradish peroxidase (HRP) enzyme. It specifically targets the Protein flightless-1 homolog (FLII), which plays crucial roles in transcriptional activation, cytoskeletal regulation, and early embryonic development. The antibody is available with reactivity against both human and mouse FLII proteins, with human-reactive variants recognizing the 577-729AA region and mouse-reactive variants targeting the 495-827AA region of the respective FLII proteins . The direct conjugation to HRP enables visualization in immunoassays without requiring a secondary antibody, streamlining experimental workflows while maintaining specificity.

What applications is FLII Antibody, HRP conjugated typically used for?

FLII Antibody, HRP conjugated has been primarily validated for Enzyme-Linked Immunosorbent Assay (ELISA) applications . While not explicitly validated for other techniques in the provided data, HRP-conjugated antibodies are generally suitable for applications including western blotting, immunohistochemistry, and immunocytochemistry. When using this antibody for direct western blotting, researchers should be aware that signal amplification may be limited compared to traditional indirect detection methods using unconjugated primary and HRP-conjugated secondary antibodies . The conjugation to HRP makes this antibody particularly valuable for reducing background signal in assays where cross-reactivity between secondary antibodies and endogenous immunoglobulins is a concern.

How should FLII Antibody, HRP conjugated be stored?

For optimal stability and performance, FLII Antibody, HRP conjugated should be aliquoted upon receipt and stored at -20°C or -80°C . Repeated freeze-thaw cycles should be strictly avoided as they can compromise both antibody binding capacity and HRP enzymatic activity. The antibody is typically supplied in a protective buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin-300 as a preservative . When handling the antibody, it's advisable to minimize exposure to light as this can reduce HRP activity over time. Working aliquots may be stored at 4°C for up to one week, but long-term storage should be at freezing temperatures.

What is the difference between FLII antibody reactivity for Mouse versus Human samples?

The key differences between mouse and human FLII antibody preparations relate to their target immunogens, amino acid recognition regions, and potential cross-reactivity profiles as shown in the comparative table below:

These differences highlight the importance of selecting the appropriate species-specific antibody variant for your experimental model to ensure optimal target recognition and specificity .

How does the lyophilization process enhance HRP-antibody conjugation for immunoassays?

This modified approach effectively reduces the reaction volume without altering the quantity of reactants, following the principle that reaction rates are proportional to the concentration of reacting molecules. Studies have demonstrated that conjugates prepared using this lyophilization-enhanced method exhibit significantly higher sensitivity in ELISA, detecting antigens at dilutions as high as 1:5000 compared to 1:25 for classical conjugation methods . Additionally, the lyophilized activated HRP demonstrates improved stability, allowing storage at 4°C for extended periods. This enhanced conjugation efficiency creates a poly-HRP structure where multiple HRP molecules bind to each antibody, amplifying signal generation without requiring secondary antibodies or additional amplification steps.

What experimental considerations should be taken when using HRP-conjugated FLII antibody for direct western blotting?

When employing HRP-conjugated FLII antibody for direct western blotting, several critical experimental considerations must be addressed to ensure optimal results. Direct western blotting using HRP-conjugated primary antibodies eliminates the signal amplification step provided by secondary antibodies, which can significantly impact detection sensitivity . This approach is only recommended when target protein expression is moderate to high or when signal amplification is not essential for detection.

For successful direct western blotting with FLII Antibody, HRP conjugated:

• Increase primary antibody concentration: Use 5-10 times higher concentration than would be used in indirect western blotting to compensate for reduced signal amplification.

• Optimize blocking conditions: Test different blocking agents (BSA, non-fat dry milk, commercial blockers) as these can significantly affect background and signal intensity with direct detection methods.

• Extend membrane incubation time: Consider extending the primary antibody incubation to overnight at 4°C to improve binding efficiency.

• Use high-sensitivity substrate: Employ enhanced chemiluminescent (ECL) substrates designed for high sensitivity to maximize signal detection.

• Control for non-specific binding: Include appropriate negative controls to verify signal specificity, as troubleshooting direct western blots can be more challenging.

• Consider alternative detection strategies for low-abundance targets: If signal is insufficient with direct detection, switch to an indirect approach using unconjugated FLII antibody with HRP-conjugated secondary antibody .

How can the sensitivity of FLII Antibody, HRP conjugated be optimized in immunoassays?

Optimizing the sensitivity of FLII Antibody, HRP conjugated in immunoassays requires a multifaceted approach focusing on reagent quality, protocol refinement, and detection system enhancement. Research has demonstrated that modifications to standard protocols can dramatically improve detection limits, with optimized conjugates capable of detecting antigens at concentrations as low as 1.5 ng .

Key strategies for sensitivity optimization include:

• Buffer optimization: Adjust buffer pH and ionic strength to create optimal conditions for antibody-antigen interaction while preserving HRP activity. Phosphate buffers at pH 7.4 with stabilizing proteins (0.1-0.5% BSA) often provide an optimal environment .

• Incubation temperature modulation: While room temperature incubations are standard, controlled comparative studies of temperature effects (4°C, room temperature, 37°C) on binding kinetics may identify optimal conditions for specific FLII epitope recognition.

• Signal amplification systems: For ultrasensitive detection, implement tyramide signal amplification (TSA) or poly-HRP systems that can enhance signal by 10-100 fold over standard detection methods.

• Substrate selection: Choose HRP substrates based on detection method requirements. For colorimetric detection, TMB offers excellent sensitivity; for chemiluminescence, enhanced luminol reagents provide superior signal-to-noise ratios with extended signal duration.

• Specialized conjugation approaches: Considering employing lyophilization-enhanced conjugation methods that have demonstrated significantly improved sensitivity in immunoassays compared to classical conjugation protocols .

• Sample preparation refinement: Optimize protein extraction and purification methods to reduce interfering compounds that may inhibit antibody binding or HRP activity.

What are the potential cross-reactivity concerns when using FLII Antibody, HRP conjugated in complex samples?

When working with FLII Antibody, HRP conjugated in complex biological samples, researchers must address several potential cross-reactivity concerns to ensure accurate and specific target detection. The polyclonal nature of the available FLII antibodies introduces inherent variability in epitope recognition, which can affect specificity in different experimental contexts .

Primary cross-reactivity considerations include:

• Species cross-reactivity: While the antibodies are validated for specific species (human or mouse), researchers working with other model organisms should perform preliminary validation studies. The evolutionary conservation of FLII protein domains may result in unintended cross-species reactivity.

• Isoform specificity: FLII can exist in multiple isoforms resulting from alternative splicing. The antibody's immunogen (spanning specific amino acid regions 577-729AA for human or 495-827AA for mouse) may recognize multiple isoforms, potentially complicating data interpretation in tissues with differential isoform expression .

• Protein family cross-reactivity: FLII belongs to the gelsolin superfamily of actin-remodeling proteins. Structural similarities with related proteins may lead to cross-reactivity, particularly in assays with less stringent conditions.

• Post-translational modifications: FLII undergoes various post-translational modifications that may alter epitope accessibility. The antibody's ability to recognize differently modified forms should be validated in the specific experimental context.

• Sample-specific matrix effects: Complex biological samples (tissue lysates, serum, etc.) contain various proteins, lipids, and other biomolecules that may non-specifically interact with the antibody or affect HRP activity. Sample-specific optimization of blocking agents and washing protocols is essential.

To address these concerns, incorporate specificity controls including:

• Pre-absorption with recombinant FLII protein to confirm signal specificity

• Western blot analysis to verify single-band detection at the expected molecular weight

• Peptide competition assays to demonstrate epitope-specific binding

• Parallel testing with alternative FLII antibodies recognizing different epitopes

How does the binding of FLII Antibody affect the functional roles of FLII in regulating cytoskeletal rearrangements?

The binding of FLII antibody to its target protein raises important mechanistic questions regarding potential interference with FLII's biological functions, particularly in cytoskeletal regulation. FLII is essential for early embryonic development and plays critical roles in regulating cytoskeletal rearrangements involved in cytokinesis and cell migration, specifically by inhibiting Rac1-dependent paxillin phosphorylation .

When studying FLII's functional roles using antibody-based approaches, researchers should consider:

• Epitope location and functional domains: The antibody's target region (577-729AA for human or 495-827AA for mouse) may overlap with functional domains involved in protein-protein interactions or enzymatic activity. Mapping the epitope relative to known functional domains is essential for interpreting functional studies.

• Neutralizing potential: Depending on the epitope recognized, the antibody may possess neutralizing activity that blocks FLII interactions with binding partners or substrates. This property could be leveraged for functional studies but must be characterized to avoid misinterpretation of results.

• Steric hindrance effects: Even non-neutralizing antibodies may induce conformational changes or create steric barriers that alter FLII's interaction with cytoskeletal components or signaling molecules, particularly in live-cell applications.

• Experimental design considerations: For functional studies, researchers should:

  • Compare results using multiple antibodies targeting different FLII epitopes

  • Include appropriate isotype controls

  • Validate findings using complementary approaches (genetic knockdown/knockout, domain mutants)

  • Consider using Fab fragments for applications where Fc-mediated effects might confound results

• Application-specific considerations: In immunoprecipitation studies targeting FLII's interaction partners in cytoskeletal regulation pathways, the antibody binding may disrupt certain protein-protein interactions, potentially leading to false-negative results for transient or weak interactions.

What controls should be included when validating results from FLII Antibody, HRP conjugated in experimental workflows?

Rigorous validation of results obtained using FLII Antibody, HRP conjugated requires a comprehensive control strategy tailored to specific experimental applications. Implementing appropriate controls ensures data reliability and facilitates accurate interpretation of experimental outcomes.

Essential controls for FLII Antibody, HRP conjugated experimental workflows include:

• Antibody specificity controls:

  • Isotype control: Include a non-targeting HRP-conjugated IgG from the same species (rabbit) to assess non-specific binding

  • Peptide competition/blocking: Pre-incubate the antibody with excess recombinant FLII protein or the immunizing peptide to confirm signal specificity

  • Knockout/knockdown validation: Compare signal between wild-type samples and those with genetically reduced FLII expression

• Technical controls:

  • Loading controls: Include housekeeping protein detection for western blotting or cell number normalization for ELISA

  • Standard curve: Generate a standard curve using recombinant FLII protein to enable quantitative analysis

  • Signal range validation: Perform serial dilutions of both antibody and sample to confirm linear detection range

• HRP conjugation controls:

  • Enzyme activity control: Include a known HRP substrate reaction without antibody to confirm enzyme activity

  • Unconjugated antibody comparison: In parallel experiments, compare results from HRP-conjugated versus unconjugated primary antibody with HRP-secondary antibody detection

• Application-specific controls:

  • For ELISA: Include blank wells (no antigen), background control wells (no primary antibody), and positive control wells (with verified FLII-expressing samples)

  • For Western blotting: Include molecular weight markers and positive control lysates with known FLII expression

  • For cell/tissue staining: Include secondary-only controls and known positive/negative tissue controls

• Cross-validation controls:

  • Orthogonal method validation: Confirm findings using an alternative detection approach (e.g., mass spectrometry, PCR)

  • Alternative antibody validation: Verify results using a different FLII antibody recognizing a distinct epitope

How can FLII Antibody, HRP conjugated be used to investigate estrogen hormone signaling pathways?

FLII Antibody, HRP conjugated offers valuable research applications for investigating estrogen hormone signaling pathways due to FLII's established role as a coactivator in transcriptional activation by hormone-activated nuclear receptors, particularly in estrogen hormone signaling . Strategic experimental approaches can leverage this antibody to elucidate FLII's specific functions in estrogen-responsive pathways.

Methodological approaches for studying estrogen signaling with FLII Antibody, HRP conjugated include:

• Estrogen-responsive transcriptional complex analysis:

  • Use chromatin immunoprecipitation (ChIP) assays with FLII antibody to identify estrogen-dependent recruitment to estrogen response elements (EREs)

  • Combine with sequential ChIP (re-ChIP) to characterize FLII co-localization with estrogen receptor (ER) and other coactivators like NCOA2 and CARM1

  • Quantify binding dynamics using ELISA-based ChIP methods leveraging the HRP conjugation for direct detection

• Hormone-dependent FLII protein interactions:

  • Develop co-immunoprecipitation protocols optimized for nuclear receptor complexes

  • Investigate interaction kinetics between FLII and estrogen receptors under various hormonal conditions

  • Quantify protein-protein interactions using modified ELISA approaches leveraging HRP conjugation

• Signaling pathway crosstalk investigation:

  • Monitor FLII involvement in non-genomic estrogen signaling by analyzing cytoskeletal rearrangements in response to estrogen treatment

  • Investigate FLII-dependent phosphorylation events downstream of estrogen signaling, focusing on paxillin and Rac1 regulation

  • Develop multiplexed assays combining FLII detection with markers of activated estrogen signaling

• Cell-type specific responses to estrogen:

  • Optimize immunohistochemistry protocols using FLII Antibody, HRP conjugated for tissue-specific analysis

  • Compare FLII expression and localization across different estrogen-responsive tissues

  • Correlate FLII expression patterns with estrogen receptor status in tissue samples

• Temporal dynamics of FLII in estrogen signaling:

  • Establish time-course experiments tracking FLII recruitment to transcriptional complexes following estrogen stimulation

  • Develop pulse-chase approaches to monitor FLII protein turnover rates in response to estrogen

What techniques can be employed to troubleshoot low signal issues with FLII Antibody, HRP conjugated?

When encountering low signal issues with FLII Antibody, HRP conjugated, a systematic troubleshooting approach is essential to identify and resolve underlying causes. Research on HRP-antibody conjugates indicates that both conjugation quality and experimental conditions significantly impact detection sensitivity .

Comprehensive troubleshooting strategy for low signal issues:

• Antibody integrity assessment:

  • Evaluate HRP enzymatic activity using a direct substrate test

  • Check storage conditions and freeze-thaw history, as repeated freeze-thaw cycles severely compromise antibody performance

  • Verify antibody expiration date and consider obtaining a new lot if activity is suspected to be compromised

• Protocol optimization:

  • Titrate antibody concentration (try 2-5 fold higher concentrations than recommended starting dilutions)

  • Extend incubation times to enhance antigen binding (overnight at 4°C for western blots or ELISAs)

  • Adjust blocking conditions to reduce non-specific binding while preserving epitope accessibility

  • Modify washing protocols to remove unbound antibody while retaining specific interactions

• Signal enhancement strategies:

  • Implement signal amplification systems compatible with HRP (e.g., tyramide signal amplification)

  • For western blotting, consider switching to an indirect detection method using unconjugated primary and HRP-conjugated secondary antibodies for signal amplification

  • Optimize substrate selection and development time (extended exposure for chemiluminescence or longer substrate development for colorimetric detection)

• Sample preparation refinement:

  • Increase target protein concentration through optimized extraction methods

  • Remove potential interfering compounds through additional purification steps

  • Check sample buffer compatibility with antibody binding and HRP activity

  • For fixed samples, optimize fixation and antigen retrieval methods to enhance epitope accessibility

• Instrumentation considerations:

  • Verify detector sensitivity settings and calibration

  • Adjust exposure time or gain settings to capture weak signals

  • Consider alternative detection instruments with higher sensitivity

• Advanced solutions for persistent issues:

  • Re-conjugate the antibody using enhanced methods like the lyophilization-based protocol that has demonstrated significantly improved sensitivity

  • Switch to alternative detection systems (e.g., from colorimetric to chemiluminescent)

  • Consider amplifying the target protein through upstream methods (overexpression systems, protein concentration)

How does buffer composition affect the stability and performance of FLII Antibody, HRP conjugated?

Buffer composition plays a critical role in maintaining the stability, activity, and specificity of FLII Antibody, HRP conjugated across various storage conditions and experimental applications. Each buffer component serves specific functions that collectively impact antibody performance and longevity.

Critical buffer components and their effects on FLII Antibody, HRP conjugated:

• Glycerol (50%):

  • Cryoprotective effect: Prevents ice crystal formation during freezing, protecting antibody structure

  • Stabilizing effect: Reduces protein denaturation by preferential hydration

  • Storage impact: Enables storage at -20°C without compromising antibody integrity

  • Application consideration: High glycerol content may require dilution before use in certain applications

• Phosphate-buffered saline (0.01M PBS, pH 7.4):

  • pH stabilization: Maintains optimal pH for both antibody binding and HRP catalytic activity

  • Ionic strength: Provides physiological salt concentration that preserves antibody conformation

  • Buffer capacity: Resists pH changes during storage and experimental procedures

  • HRP compatibility: Supports enzymatic activity while minimizing auto-oxidation

• Preservatives (0.03% Proclin-300):

  • Antimicrobial effect: Prevents bacterial and fungal contamination during storage

  • Enzyme protection: Selected for compatibility with HRP activity (unlike sodium azide, which inhibits HRP)

  • Long-term stability: Enables extended storage without activity loss

  • Safety consideration: Less toxic alternative to traditional preservatives

• Experimental buffer modifications:

  • Reducing agents: Must be avoided as they can disrupt disulfide bonds in antibodies and inactivate HRP

  • Detergents: Low concentrations (0.05-0.1% Tween-20) can reduce non-specific binding without affecting HRP activity

  • Protein additives: BSA (0.1-1%) can enhance stability and reduce non-specific interactions in working dilutions

  • Metal chelators: EDTA should be used cautiously as it may affect HRP activity, which requires heme-bound iron

• Performance optimization through buffer adjustment:

  • For ELISA applications: Consider adding 0.05% Tween-20 to washing buffers to reduce background

  • For concentrated storage: The current 50% glycerol formulation is optimal for freeze protection

  • For dilute working solutions: Add 0.5-1% BSA to dilution buffers to prevent adsorption to plastic surfaces and enhance stability

  • For extended working sessions: Prepare fresh working dilutions in buffers containing stabilizing proteins

What considerations should be made when optimizing incubation times for FLII Antibody, HRP conjugated in different applications?

Optimizing incubation times for FLII Antibody, HRP conjugated requires balancing antibody-antigen binding kinetics, HRP enzyme stability, and practical experimental constraints. Different applications demand tailored approaches to maximize signal-to-noise ratio while maintaining experimental efficiency.

Application-specific incubation time considerations: