SERPINF1 Antibody, Biotin conjugated

What is SERPINF1 and what are its key biological functions?

SERPINF1, also known as Pigment Epithelium-Derived Factor (PEDF), is a multifunctional secreted protein with anti-angiogenic, anti-tumorigenic, and neurotrophic properties. Despite belonging to the serpin family, it does not undergo the standard S (stressed) to R (relaxed) conformational transition characteristic of active serpins and therefore exhibits no serine protease inhibitory activity .

Its key functions include:

• Inducing extensive neuronal differentiation in retinoblastoma cells

• Potently inhibiting angiogenesis

• Suppressing retinal neovascularization and endothelial cell proliferation

• Inhibiting both VEGFR-1 and VEGFR-2, contributing to its antiangiogenic function

• Inhibiting cancer cell proliferation and increasing apoptosis via the FAS/FASL pathway

SERPINF1 expression begins early in human development, with detection in the human retina at 7.4 weeks of gestation, suggesting a role in retinal neuron differentiation .

What are the structural characteristics of SERPINF1 Antibody, Biotin conjugated?

SERPINF1 Antibody, Biotin conjugated typically consists of:

• Host Species: Predominantly rabbit-derived polyclonal antibodies

• Target: Human, rat, or mouse SERPINF1 protein (species reactivity varies by product)

• Conjugation: Biotin molecule covalently attached to facilitate detection in various assay systems

• Formulation: Generally supplied in liquid form with stabilizing buffers

• Buffer Composition: Typically contains preservatives (e.g., 0.03% Proclin 300), 50% glycerol, and PBS at pH 7.4

• Molecular Weight: Targets SERPINF1, which has a calculated molecular weight of approximately 46 kDa

The biotin conjugation enables the antibody to be detected using streptavidin-based detection systems, enhancing sensitivity in various experimental applications.

How should SERPINF1 Antibody, Biotin conjugated be optimally diluted for different experimental applications?

Optimal dilution ratios vary by application and specific antibody preparation. Based on available research products, the following dilution ranges are recommended as starting points:

The actual working concentration varies and should be determined by the researcher through titration experiments. Always prepare fresh dilutions and mix thoroughly before use to ensure consistent results .

What is the recommended methodology for sample preparation when using SERPINF1 Antibody, Biotin conjugated in ELISA?

For optimal ELISA results with SERPINF1 Antibody, Biotin conjugated:

• Buffer Selection: For antibody dilution, use a standardized antibody diluent buffer that maintains protein stability and minimizes background .

• Sample Preparation:

  • Cell Culture Supernatants: Centrifuge to remove particulates

  • Cell Lysates: Use appropriate lysis buffers compatible with the ELISA kit components

  • Serum Samples: Dilute appropriately (typically 1:50 to 1:500) depending on expected SERPINF1 concentration

  • Plasma Samples: Heparin or EDTA-treated samples are preferred; avoid citrate-plasma as it may interfere with the assay

• Antibody Preparation:

  • Dilute the biotinylated anti-SERPINF1 antibody 1:100 with antibody diluent buffer

  • Mix thoroughly to ensure homogeneity

  • Prepare fresh dilutions for each experiment

• Detection System:

  • Use Avidin-Biotin-Peroxidase Complex for detection

  • Follow with appropriate HRP substrate (e.g., TMB) for visualization

  • Add acidic stop solution to convert blue reaction product to yellow for quantification

How can researchers troubleshoot inconsistent results in ELISA experiments using SERPINF1 Antibody, Biotin conjugated?

Inconsistent ELISA results may stem from several factors. Here's a systematic approach to troubleshooting:

• Antibody Stability Issues:

  • Avoid repeated freeze-thaw cycles that can degrade antibody quality

  • Store antibody at recommended temperatures (-20°C or -80°C for long-term; 4°C for up to one month for frequent use)

  • Check expiration dates and visible signs of degradation (precipitation, color changes)

• Technical Variables:

  • Ensure consistent incubation times and temperatures across experiments

  • Verify complete washing between steps to remove unbound reagents

  • Confirm proper plate sealing during incubations to prevent edge effects

  • Use calibrated pipettes for accurate volume dispensing

• Sample-Related Factors:

  • Test for matrix effects by performing dilution linearity experiments

  • Ensure samples remain within the assay's linear range (prepare multiple dilutions)

  • Check for potential interfering substances in complex biological samples

• Optimization Strategies:

  • Perform antibody titration to determine optimal concentration for your specific samples

  • Include appropriate positive and negative controls in each assay

  • Consider examining fresh vs. frozen samples to assess stability impact

If problems persist, verify antibody reactivity with your sample species, as some SERPINF1 antibodies show restricted species reactivity (e.g., human-only or rat-specific) .

What are the key considerations when analyzing cross-reactivity data for SERPINF1 Antibody, Biotin conjugated?

When analyzing cross-reactivity data, researchers should consider:

• Species Homology Assessment:

  • Compare sequence homology of the immunogen region across species

  • Different antibody products target different epitopes (e.g., amino acids 128-418 in humans vs. 78-121 in rats )

  • Some antibodies are raised against specific domains or peptides (e.g., antibody A02034T307 targets amino acids 280-320 )

• Validation Methods Evaluation:

  • Examine the methods used to validate cross-reactivity (WB, IHC, ELISA)

  • Consider whether validation was performed using endogenous protein or recombinant samples

  • Assess positive controls used in validation studies

• Cross-Reactivity Matrix Analysis:

• Factors Affecting Cross-Reactivity:

  • Post-translational modifications may differ between species

  • Tissue-specific expression patterns could influence detection

  • Sample preparation methods may affect epitope accessibility

When interpreting cross-reactivity results, consider that predicted reactivity may require experimental verification in your specific experimental system.

How can SERPINF1 Antibody, Biotin conjugated be utilized in multiplex immunoassays for studying angiogenesis pathways?

SERPINF1 Antibody, Biotin conjugated offers several advantages in multiplex angiogenesis pathway studies:

• Multiplex Platform Integration:

  • The biotin conjugation enables incorporation into streptavidin-based detection systems commonly used in multiplex platforms

  • Researchers can pair this antibody with differently labeled antibodies targeting other angiogenesis factors (VEGF, angiopoietins, etc.)

• Experimental Design Considerations:

  • Validate antibody performance in single-analyte assays before incorporating into multiplex systems

  • Test for potential cross-reactivity with other biotinylated antibodies in your panel

  • Optimize signal-to-noise ratios by adjusting antibody concentrations

• Sample Preparation Strategy:

  • For cell culture models: Consider collecting conditioned media at multiple timepoints to track SERPINF1 secretion dynamics

  • For tissue samples: Optimize extraction protocols to simultaneously preserve SERPINF1 and other angiogenic factors

• Analysis Approach:

  • Normalize SERPINF1 levels against housekeeping proteins or total protein content

  • Consider ratio analyses between pro-angiogenic (VEGF) and anti-angiogenic (SERPINF1) factors

  • Implement statistical methods appropriate for multiplex data with multiple interdependent variables

This approach enables researchers to study the balance between pro-angiogenic and anti-angiogenic factors in complex biological systems, offering insights into angiogenesis regulation in development, cancer, and other pathological conditions .

What are the experimental considerations for studying SERPINF1's dual role in neuronal differentiation and angiogenesis inhibition?

Investigating SERPINF1's dual functionality requires careful experimental design:

• Cell System Selection:

  • Neuronal Differentiation: Utilize retinoblastoma cell lines or neural progenitor cells that respond to SERPINF1's neurotrophic effects

  • Angiogenesis Inhibition: Employ endothelial cell models (HUVECs, HMVECs) or ex vivo angiogenesis assays

• Functional Domain Analysis:

  • SERPINF1 contains distinct domains responsible for its various functions

  • Consider using domain-specific antibodies to distinguish between effects

  • The antibody's immunogen region (e.g., amino acids 78-121 or 128-418 ) may influence which function is detected

• Experimental Readouts:

  • Neuronal Differentiation Markers: βIII-tubulin, MAP2, neurite outgrowth measurements

  • Angiogenesis Inhibition Metrics: Endothelial tube formation, migration, proliferation assays

• Specialized Assay Design:

  • Co-culture systems combining neuronal and endothelial cells can reveal contextual effects

  • Time-course experiments to determine temporal aspects of each function

  • Concentration-dependent studies to identify potential threshold effects

• Control Experiments:

  • Include function-blocking experiments (using non-conjugated antibodies) to validate specific pathways

  • Compare effects of recombinant SERPINF1 with endogenous protein detection using the biotin-conjugated antibody

By carefully designing experiments that can distinguish between these functions, researchers can better understand how SERPINF1 coordinates tissue development and homeostasis through its pleiotropic activities .

How does species-specific sequence variation in SERPINF1 impact antibody selection for comparative studies?

SERPINF1 sequence variations across species significantly impact antibody selection:

• Epitope Conservation Analysis:

  • Examine the specific immunogen region used to generate the antibody

  • Some antibodies target conserved regions (higher cross-reactivity), while others target species-specific regions

  • Example: Antibody A34482 was generated against rat SERPINF1 amino acids 78-121 , while A02034T307 targets human SERPINF1 amino acids 280-320

• Species Validation Status:

  • Verify which species have been experimentally validated versus those with predicted reactivity

  • Antibody DF6547 has experimentally validated reactivity with human, mouse, and rat samples, with predicted reactivity to pig, bovine, horse, sheep, rabbit, and dog samples

• Application-Specific Considerations:

  • An antibody may cross-react in one application (e.g., ELISA) but not in others (e.g., IHC)

  • Application success depends on epitope accessibility in different assay conditions

• Experimental Approaches for Comparative Studies:

  • For multi-species studies, consider using antibodies targeting highly conserved regions

  • Validate each antibody in each species before conducting comparative analyses

  • When possible, use multiple antibodies targeting different epitopes to increase confidence in results

Understanding these factors helps researchers select appropriate antibodies for comparative studies across species and avoid misinterpretation of results due to species-specific variations in antibody recognition .

What methodological approaches can researchers use to validate SERPINF1 Antibody, Biotin conjugated in non-traditional model organisms?

When validating SERPINF1 Antibody, Biotin conjugated in non-traditional model organisms:

• Sequence Homology Assessment:

  • Perform bioinformatic analysis comparing the immunogen sequence with the target organism's SERPINF1 sequence

  • Calculate percent identity and similarity, focusing on the specific epitope region

  • Predict potential cross-reactivity based on conserved structural domains

• Stepwise Validation Protocol:

  • Initial Screening: Begin with ELISA using recombinant protein if available

  • Western Blot Validation: Confirm antibody detects a protein of the expected molecular weight (approximately 46 kDa)

  • Tissue Expression Profiling: Compare detection patterns with known SERPINF1 expression in well-characterized species

  • Functional Validation: Confirm biological activity of the detected protein through knockdown or blocking experiments

• Controls Integration:

  • Include positive controls from validated species (e.g., human or rat samples)

  • Implement negative controls using pre-immune serum or isotype-matched irrelevant antibodies

  • Consider using tissues from SERPINF1 knockout models (if available) as definitive negative controls

• Optimization Strategies:

  • Test multiple fixation and antigen retrieval methods for IHC applications

  • Adjust antibody concentration through systematic titration experiments

  • Modify incubation conditions (time, temperature, buffer composition) to enhance specific binding

• Confirmation Approaches:

  • Validate findings using an independent method (e.g., mass spectrometry)

  • Compare results with multiple antibodies targeting different SERPINF1 epitopes

  • Correlate antibody detection with mRNA expression data