SERPINF1 Antibody

What is SERPINF1 and why is it important for research?

SERPINF1, also known as PEDF (Pigment Epithelium-Derived Factor), is a 46-50 kDa glycoprotein member of the serpin superfamily of protease inhibitors . Despite belonging to the serpin family, SERPINF1 is considered non-protease inhibiting and instead possesses potent antiangiogenic and neurotrophic activities . SERPINF1 is expressed by diverse cell types including retinal pigment epithelium, breast epithelium, fibroblasts, astrocytes, and hepatocytes . It circulates in blood and binds to type I collagen plus heparan sulfate .

The protein contains several functional domains including a nuclear localization signal (aa 146-149), a neuroprotective motif (aa 354-359), and an antiangiogenesis segment (aa 387-411) . Its multifunctional nature makes it relevant for research in various fields including cancer biology, neurodegenerative diseases, and metabolic disorders due to its roles in regulating angiogenesis, cell differentiation, and neuroprotection .

What are the common applications for SERPINF1 antibodies in basic research?

SERPINF1 antibodies are widely utilized in several applications:

• Western Blot (WB): Detection of SERPINF1 in cell lysates and tissue samples, with typical bands observed at approximately 46-50 kDa . This technique helps quantify expression levels across different experimental conditions.

• Immunohistochemistry (IHC): Visualization of SERPINF1 distribution in tissue sections, with specific staining observed in structures like convoluted tubules in human kidney samples .

• Immunofluorescence/Immunocytochemistry (IF/ICC): Determination of subcellular localization and expression patterns in cultured cells .

• ELISA: Quantitative measurement of SERPINF1 levels in serum, plasma, or cell culture supernatants .

Each application requires specific optimization of antibody dilutions, typically 1:500-1:1000 for Western blot applications and 15 μg/mL for immunohistochemistry .

How do I select the appropriate SERPINF1 antibody for my specific research question?

Selection of the optimal SERPINF1 antibody should be based on several critical factors:

• Species reactivity: Match the antibody reactivity with your experimental model. Available antibodies show reactivity with human, mouse, and rat samples, with predicted cross-reactivity for other species like pig, bovine, and sheep .

• Application compatibility: Verify that the antibody has been validated for your specific application (WB, IHC, IF, ELISA) .

• Epitope recognition: Consider the specific region of SERPINF1 targeted by the antibody. Some antibodies recognize epitopes within amino acids 141-240 of human SERPINF1, while others target different regions . This is particularly important if studying specific domains or post-translational modifications.

• Clonality: Polyclonal antibodies offer broad epitope recognition and stronger signals, while monoclonal antibodies provide higher specificity. Both types are available for SERPINF1 detection .

• Validation data: Review available data showing specificity in relevant tissues. For example, some SERPINF1 antibodies have been validated in human hepatocellular carcinoma cell lines, human liver tissue, and mouse embryonal carcinoma cells .

What are the optimal conditions for Western blot detection of SERPINF1?

For optimal Western blot detection of SERPINF1, follow these evidence-based recommendations:

• Sample preparation:

  • For cell lysates: Use reducing conditions and immunoblot buffer (e.g., Immunoblot Buffer Group 1) .

  • For secreted SERPINF1: Collect 15μl of conditioned media for electrophoresis .

• Gel electrophoresis:

  • Use a 12-230 kDa separation system for optimal resolution .

  • PVDF membranes are recommended for protein transfer .

• Blocking and antibody incubation:

  • Block membranes with 5% milk in Tris buffered saline/0.1% Tween (TBST) .

  • Use antibody dilutions of 0.2-2 μg/mL for goat anti-human/mouse SERPINF1 antibodies .

  • For rabbit polyclonal antibodies, recommended dilution ranges are 1:500-1:1000 .

• Detection:

  • Use appropriate HRP-conjugated secondary antibodies matched to the primary antibody host species .

  • Detect signals using chemiluminescence detection systems .

• Expected results:

  • SERPINF1 typically appears as a specific band at approximately 46-50 kDa .

  • In some systems (e.g., Simple Western), the protein may migrate slightly differently (around 56 kDa) .

How should I optimize immunohistochemistry protocols for SERPINF1 detection in different tissue types?

Successful immunohistochemical detection of SERPINF1 requires tissue-specific optimization:

• Tissue preparation:

  • Use immersion-fixed, paraffin-embedded tissue sections .

  • Standard deparaffinization and antigen retrieval techniques should be employed.

• Antibody concentration and incubation:

  • Start with 15 μg/mL of antibody for paraffin sections .

  • Incubate overnight at 4°C for optimal results .

• Detection systems:

  • For chromogenic detection, HRP-DAB systems work effectively (e.g., Anti-Goat HRP-DAB Cell & Tissue Staining Kit) .

  • Counterstain with hematoxylin for contrast .

• Tissue-specific considerations:

  • In kidney tissue, expect specific staining in convoluted tubules .

  • For other tissues, optimization may be required as expression patterns vary.

• Controls:

  • Include positive control tissues known to express SERPINF1 (human liver, mouse liver, rat liver) .

  • Include negative controls (omitting primary antibody) to confirm specificity.

• Signal interpretation:

  • SERPINF1 can be localized to both intracellular and secreted compartments, so staining patterns should be interpreted accordingly.

What are the best methods for quantifying SERPINF1 in biological fluids?

For accurate quantification of SERPINF1 in biological fluids:

• ELISA-based methods:

  • Commercial human PEDF ELISA kits offer standardized protocols for serum analysis .

  • Follow manufacturer recommendations for sample dilution and assay conditions.

  • Collect blood samples and process immediately to prevent degradation (freeze at -80°C until analysis) .

• Western blot quantification:

  • Use densitometry analysis of Western blot bands compared against standard curves of recombinant protein .

  • Include loading controls and normalization standards.

• Other quantitative methods:

  • Simple Western™ systems provide automated, quantitative analysis with high reproducibility .

  • Mass spectrometry-based approaches may be used for absolute quantification in complex samples.

• Sample handling considerations:

  • Collect blood retro-orbitally or through other established methods .

  • Process samples immediately and store at -80°C to preserve protein integrity .

  • Avoid repeated freeze-thaw cycles to prevent protein degradation .

How can SERPINF1 antibodies be used to investigate protein-protein interactions and signaling pathways?

Advanced applications for studying SERPINF1 interactions include:

• Co-immunoprecipitation (Co-IP):

  • Use SERPINF1 antibodies to pull down protein complexes from cell lysates.

  • Analyze binding partners through subsequent Western blot or mass spectrometry.

  • This approach can help identify interactions with receptors, extracellular matrix components like collagen, or heparan sulfate .

• Chromatin immunoprecipitation (ChIP):

  • SERPINF1 contains a nuclear localization signal (aa 146-149) , suggesting potential nuclear functions.

  • ChIP using SERPINF1 antibodies can identify DNA binding sites or chromatin associations.

• Proximity ligation assay (PLA):

  • This technique can visualize and quantify SERPINF1 interactions with other proteins in situ at single-molecule resolution.

  • Particularly useful for confirming interactions identified through other methods.

• Phospho-specific analysis:

  • SERPINF1 undergoes phosphorylation at multiple sites that affect bioactivity .

  • Phospho-specific antibodies or general phospho-detection after immunoprecipitation can reveal activation states.

• Functional blocking studies:

  • Some antibodies may block SERPINF1's functional domains (neuroprotective motif or antiangiogenesis segment) .

  • This approach can help delineate specific functions in biological processes.

What experimental approaches are effective for studying the role of SERPINF1 in angiogenesis and tumor development?

To investigate SERPINF1's role in angiogenesis and tumor biology:

• In vitro angiogenesis assays:

  • Tube formation assays using endothelial cells treated with recombinant SERPINF1 or conditioned media from cells with modified SERPINF1 expression.

  • Measure inhibition of endothelial cell migration, proliferation, and tube formation.

  • Use SERPINF1 antibodies to neutralize activity or confirm expression levels .

• In vivo tumor models:

  • Generate xenograft models with tumors expressing different levels of SERPINF1.

  • Use viral vector systems (e.g., helper-dependent adenoviral vectors) for SERPINF1 delivery .

  • Monitor tumor growth, vascularization, and metastasis.

  • Confirm SERPINF1 expression using antibody-based detection in tumor sections .

• Transgenic models:

  • Utilize existing Serpinf1-/- mouse models for restoration studies .

  • Introduce SERPINF1 expression using liver-specific promoters (e.g., PEPCK) .

  • Monitor serum levels via ELISA and functional outcomes .

• 3D culture systems:

  • Develop spheroid or organoid cultures with varying SERPINF1 expression.

  • Analyze vascular mimicry, invasion, and other tumor-related phenotypes.

  • Use immunohistochemistry with SERPINF1 antibodies to confirm expression patterns .

How can I effectively investigate post-translational modifications of SERPINF1 using available antibodies?

For studying post-translational modifications (PTMs) of SERPINF1:

• Phosphorylation analysis:

  • SERPINF1 undergoes phosphorylation at multiple sites that affect its bioactivity .

  • Use phospho-specific antibodies if available, or:

  • Immunoprecipitate SERPINF1 using general antibodies, then detect phosphorylation with anti-phospho antibodies.

  • Combine with phosphatase treatments as controls.

• Glycosylation studies:

  • SERPINF1 is a glycoprotein of approximately 50 kDa .

  • Compare migration patterns before and after deglycosylation enzyme treatment.

  • Use lectins in combination with SERPINF1 antibodies to characterize glycosylation patterns.

• Proteolytic processing:

  • Investigate potential cleavage products using antibodies targeting different epitopes.

  • Compare full-length and processed forms across different tissues and conditions.

  • N-terminal versus C-terminal antibodies can help identify processing events.

• Immunoprecipitation followed by mass spectrometry:

  • Use SERPINF1 antibodies to enrich the protein from complex samples.

  • Perform mass spectrometry analysis to identify and map various PTMs.

  • This approach can reveal unexpected modifications and their stoichiometry.

How can I troubleshoot common issues with SERPINF1 antibody-based detection methods?

When encountering detection problems with SERPINF1 antibodies:

• Weak or no signal in Western blot:

  • Increase antibody concentration (try 0.2-2 μg/mL range for Western blot) .

  • Extend incubation time to overnight at 4°C.

  • For secreted SERPINF1, concentrate culture media before loading .

  • Ensure reducing conditions are used for sample preparation .

  • Check if the antibody epitope matches your species of interest .

• High background in immunohistochemistry:

  • Optimize blocking (5% milk or BSA in TBST recommended) .

  • Dilute antibody further (start with 15 μg/mL and adjust as needed) .

  • Increase washing steps and duration.

  • Use more specific detection systems with lower cross-reactivity.

• Inconsistent results between experiments:

  • Standardize sample collection and processing (immediate freezing at -80°C recommended) .

  • Avoid repeated freeze-thaw cycles of samples .

  • Use recombinant protein standards for quantitative comparisons.

  • Ensure consistent experimental conditions (same cells passage, treatment timing).

• Multiple bands in Western blot:

  • Verify if additional bands represent modified forms (glycosylated vs. non-glycosylated).

  • Check for proteolytic processing (SERPINF1 can undergo cleavage).

  • Increase antibody specificity by further dilution.

  • Use positive control samples (human liver, mouse liver) .

How do I interpret different SERPINF1 expression patterns across various tissues and cell types?

For proper interpretation of SERPINF1 expression patterns:

• Tissue-specific expression considerations:

  • SERPINF1 is expressed by diverse cell types including retinal pigment epithelium, breast epithelium, fibroblasts, astrocytes, and hepatocytes .

  • In kidney, expect specific staining in convoluted tubules .

  • Liver typically shows strong expression and serves as a good positive control .

  • Consider both cellular and extracellular matrix localization due to SERPINF1's secreted nature .

• Subcellular localization interpretation:

  • Despite being primarily secreted, SERPINF1 contains a nuclear localization signal (aa 146-149) .

  • It can be found in melanosomes and secretory pathways .

  • Different antibodies may preferentially detect specific subcellular pools.

• Quantitative comparison methodologies:

  • Use standardized loading controls appropriate for each sample type.

  • For secreted forms, normalize to total protein or another constitutively secreted protein.

  • Consider both intracellular and secreted fractions for complete expression analysis.

• Distinguishing specific from non-specific staining:

  • Always include negative controls (secondary antibody only).

  • Verify patterns with multiple antibodies targeting different epitopes when possible.

  • Compare staining patterns with published literature for consistency.

What considerations are important when comparing results from different SERPINF1 antibodies?

When using multiple antibodies or comparing results across studies:

• Epitope differences:

  • Different antibodies target distinct regions of SERPINF1 (e.g., within aa 141-240 or other segments) .

  • Some epitopes may be masked by protein interactions or conformational changes.

  • Functional domains (neuroprotective motif aa 354-359 or antiangiogenesis segment aa 387-411) may be differentially accessible .

• Clonality effects:

  • Polyclonal antibodies provide broader epitope recognition but potentially lower specificity .

  • Monoclonal antibodies offer higher specificity but may miss some protein variants.

  • Consider using both types for comprehensive analysis.

• Host species considerations:

  • Antibodies from different host species (rabbit, goat) may perform differently in certain applications .

  • Secondary antibody selection must match the host species to avoid detection issues.

• Cross-reactivity profiles:

  • Verify species cross-reactivity experimentally even if predicted (e.g., antibodies may react with human, mouse, and rat, but predicted reactivity for pig, bovine, etc. should be validated) .

  • Different antibodies may have distinct cross-reactivity profiles.

• Standardization approaches:

  • Use recombinant SERPINF1 protein as a universal standard across experiments.

  • Run side-by-side comparisons when changing antibodies.

  • Document exact protocols for reproducibility.

How can I design experiments to study the dual neurotrophic and antiangiogenic functions of SERPINF1?

For investigating SERPINF1's dual functionality:

• Domain-specific functional analysis:

  • Design experiments targeting the neuroprotective motif (aa 354-359) versus the antiangiogenesis segment (aa 387-411) .

  • Use domain-specific antibodies or blocking peptides to inhibit specific functions.

  • Generate recombinant proteins with mutations in specific functional domains.

• Cell type-specific responses:

  • Compare effects on neuronal cells (for neurotrophic activity) versus endothelial cells (for antiangiogenic activity).

  • Use conditioned media from cells expressing SERPINF1 (e.g., stable MC3T3 cell lines expressing SERPINF1) .

  • Measure neurite outgrowth, cell survival, and differentiation in neuronal models.

  • Assess tube formation, migration, and proliferation in endothelial models.

• In vivo models for dual function assessment:

  • Utilize models that allow simultaneous observation of neural and vascular effects.

  • For example, retina models where both neuronal protection and vascular development can be monitored.

  • Use viral vector systems (e.g., HDAd-SERPINF1) for targeted expression .

• Receptor-based mechanisms:

  • Investigate different receptors mediating neurotrophic versus antiangiogenic effects.

  • Use antibodies to confirm SERPINF1 binding to specific receptors via co-immunoprecipitation.

  • Perform receptor blocking studies to differentiate pathway-specific effects.

What are effective strategies for restoring SERPINF1 expression in disease models?

Based on the literature, effective SERPINF1 restoration approaches include:

• Viral vector-based delivery systems:

  • Helper-dependent adenoviral (HDAd) vectors expressing SERPINF1 under tissue-specific promoters (e.g., liver-restricted PEPCK promoter) .

  • Lentiviral systems for stable expression in cell models .

  • Dose optimization is critical (5×10¹¹VP/kg to 5×10¹²VP/kg) to achieve therapeutic serum levels .

• Cell-based delivery strategies:

  • Generate stably expressing cell lines (e.g., MC3T3 cells expressing SERPINF1) .

  • Confirm expression via Western blot of conditioned media .

  • Use in transplantation or co-culture experiments.

• Monitoring restoration efficacy:

  • Measure serum PEDF by ELISA to confirm successful expression .

  • Verify gene expression in target tissues using real-time PCR .

  • Assess functional outcomes specific to the disease model (e.g., bone parameters in osteogenesis imperfecta models) .

• Long-term expression considerations:

  • SERPINF1 expression can be detected in liver tissue 5 months after viral injection .

  • Design experiments with appropriate time courses to capture both acute and chronic effects.

  • Include multiple timepoints for sample collection and analysis.

How can I design experiments to investigate contradictory findings about SERPINF1 function across different disease contexts?

To address contradictory findings about SERPINF1:

• Context-dependent experimental design:

  • Compare identical SERPINF1 interventions across multiple cell types and disease models.

  • Control for microenvironmental factors that may modify SERPINF1 activity.

  • Investigate post-translational modifications that could alter function in different contexts .

• Concentration-dependent effects:

  • Perform detailed dose-response studies to identify potential biphasic effects.

  • Compare physiological versus supraphysiological concentrations.

  • Use dose-controlled viral delivery systems (e.g., 5×10¹¹VP/kg versus 5×10¹²VP/kg) .

• Temporal dynamics analysis:

  • Study acute versus chronic effects of SERPINF1 restoration.

  • Include multiple timepoints (1 week, 2 weeks, 5 months) for comprehensive evaluation .

  • Consider adaptive responses that may develop over time.

• Receptor and signaling pathway dissection:

  • Identify receptor engagement in different tissues and disease states.

  • Use pathway inhibitors to determine if signaling divergence explains contextual differences.

  • Perform phosphoproteomic analysis to map activated pathways comprehensively.

• Combined in vitro and in vivo approaches:

  • Validate cell culture findings in appropriate animal models.

  • Use Serpinf1-/- mice as platforms for restoration studies .

  • Employ tissue-specific expression systems to isolate effects.

What are the storage and handling recommendations for maximizing SERPINF1 antibody performance?

For optimal antibody performance and longevity:

• Storage conditions:

  • Store antibodies at -20 to -70°C for long-term storage (up to 12 months) .

  • For short-term storage (up to 1 month), keep at 2-8°C under sterile conditions after reconstitution .

  • For medium-term storage (up to 6 months), maintain at -20 to -70°C under sterile conditions after reconstitution .

• Reconstitution protocols:

  • Use appropriate reconstitution calculators or manufacturer guidelines .

  • Some antibodies are supplied in storage buffers containing preservatives (e.g., 0.03% Proclin 300) and stabilizers (50% Glycerol, 0.01M PBS, pH 7.4) .

• Aliquoting recommendations:

  • Prepare small aliquots to avoid repeated freeze-thaw cycles .

  • Use sterile techniques when handling reconstituted antibodies.

• Quality control measures:

  • Test antibody performance periodically with positive control samples.

  • Monitor for any changes in specificity or sensitivity over time.

  • Record lot numbers and performance characteristics for reproducibility.

How can emerging technologies enhance SERPINF1 research beyond traditional antibody applications?

Innovative approaches for advancing SERPINF1 research include:

• Single-cell analysis techniques:

  • Apply single-cell RNA-seq to identify cell populations expressing SERPINF1 in heterogeneous tissues.

  • Use mass cytometry (CyTOF) with metal-conjugated SERPINF1 antibodies for high-dimensional phenotyping.

  • Spatially resolved transcriptomics to map SERPINF1 expression in tissue contexts.

• CRISPR-based approaches:

  • Generate knock-in reporter lines to monitor SERPINF1 expression in live cells.

  • Create domain-specific mutations to dissect function with precision.

  • Develop CRISPR activation/inhibition systems for controlled expression studies.

• Advanced imaging methods:

  • Super-resolution microscopy to visualize SERPINF1 localization at subcellular resolution.

  • Intravital imaging with fluorescently tagged antibodies to track dynamics in vivo.

  • FRET-based sensors to monitor SERPINF1 interactions with binding partners.

• Protein engineering approaches:

  • Develop recombinant SERPINF1 variants with enhanced stability or function.

  • Create bifunctional SERPINF1 fusion proteins for targeted delivery.

  • Generate antibody-drug conjugates targeting SERPINF1-expressing cells.

What methodological considerations are important when studying SERPINF1 in specialized research areas?

For specialized SERPINF1 research applications:

• Bone and skeletal research:

  • Combine SERPINF1 antibody detection with mineralization assays (Alizarin red staining) .

  • Consider both direct effects on osteoblasts and indirect effects via angiogenesis.

  • Use Serpinf1-/- mouse models for restoration studies in osteogenesis imperfecta .

• Ocular research:

  • SERPINF1 was originally identified in retinal pigment epithelium .

  • Use specialized tissue preservation methods to maintain eye tissue integrity.

  • Combine with markers of retinal neurons and vasculature for comprehensive analysis.

• Cancer research:

  • Account for heterogeneity of SERPINF1 expression within tumors.

  • Correlate with markers of angiogenesis, invasion, and metastasis.

  • Consider paradoxical effects in different cancer types and stages.

• Neurodegenerative disease models:

  • Focus on the neuroprotective motif (aa 354-359) of SERPINF1 .

  • Combine with markers of neuronal health, synaptic function, and neuroinflammation.

  • Consider blood-brain barrier penetration issues for therapeutic applications.