PEDF Antibody

What is PEDF and why is it significant in biomedical research?

PEDF is a 50kDa glycoprotein belonging to the serine protease inhibitor (Serpin) superfamily, though it lacks protease inhibitory activity. It is predominantly secreted by retinal pigment epithelium cells and plays crucial roles in neuroprotection and differentiation, making it vital for maintaining retinal health . PEDF inhibits angiogenesis and cell proliferation through multiple mechanisms, including inducing apoptosis in proliferating endothelial cells and inhibiting the activity of angiogenic factors such as VEGF and FGF-2 . Its neuroprotective effects operate through suppression of neuronal apoptosis induced by peroxide, glutamate, or other neurotoxins . Strategic localization of PEDF within the interphotoreceptor matrix allows effective influence on the underlying neural retina, enhancing its neuroprotective effects . These multifaceted biological activities position PEDF as a significant target in research involving neurodegenerative conditions, ocular diseases, and cancer.

What types of PEDF antibodies are available for research applications?

Based on current research tools, PEDF antibodies are available in several formats with distinct characteristics:

Monoclonal antibodies like D-10 are available in both non-conjugated and various conjugated forms, including agarose, horseradish peroxidase (HRP), phycoerythrin (PE), fluorescein isothiocyanate (FITC), and multiple Alexa Fluor® conjugates . These conjugated forms eliminate the need for secondary antibodies in certain applications. Polyclonal antibodies typically offer advantages in applications requiring higher sensitivity through recognition of multiple epitopes .

How should researchers validate the specificity of PEDF antibodies?

Antibody validation is crucial for ensuring experimental reproducibility. For PEDF antibodies, multiple complementary approaches should be employed:

• Western blot analysis using positive control tissues/cells known to express PEDF (e.g., HepG2 cells) and comparing band patterns with predicted molecular weights (41-46 kDa) . The presence of glycosylation can cause slight variations in observed molecular weight.

• Peptide competition assays using the specific immunogen peptide (e.g., human PEDF AA range:258-307) to confirm signal specificity.

• RNA interference or CRISPR knockout of PEDF in appropriate cell models, with subsequent immunoblotting to confirm signal reduction.

• Immunohistochemistry on tissues with known PEDF expression patterns (e.g., retinal pigment epithelium, mouse eye tissue) alongside negative controls (primary antibody omission, isotype controls).

• Cross-validation using multiple antibodies targeting different PEDF epitopes to confirm consistent localization patterns.

This systematic validation approach ensures that experimental observations genuinely reflect PEDF biology rather than antibody artifacts.

What are the optimal parameters for Western blot detection of PEDF?

Optimizing Western blot protocols for PEDF detection requires attention to several technical details:

For sample preparation:

• Add protease inhibitors to prevent PEDF degradation during extraction

• For secreted PEDF, concentrate conditioned media using TCA precipitation or centrifugal filters

• Load adequate protein (20-30 μg for cell lysates, as demonstrated with HepG2 samples)

For electrophoresis and transfer:

• Use 10% SDS-PAGE gels for optimal resolution of PEDF (41-46 kDa range)

• Semi-dry transfer at 15V for 30 minutes or wet transfer at 100V for 1 hour in 20% methanol transfer buffer

For antibody incubation and detection:

• Block with 5% non-fat milk in TBST for 1 hour at room temperature

• Dilute primary PEDF antibodies appropriately (e.g., 1/1000 for ab227295 , 0.1-0.2 μg/mL for polyclonal antibodies )

• Incubate overnight at 4°C with gentle rocking

• After thorough washing, apply appropriate secondary antibodies conjugated to HRP or fluorescent labels

• The detection limit for recombinant human PEDF is typically 1.5-3.0 ng/lane under either reducing or non-reducing conditions

These parameters should be optimized for each specific PEDF antibody and experimental system.

How can researchers optimize immunohistochemical detection of PEDF in tissue sections?

Successful immunohistochemical detection of PEDF requires attention to fixation, antigen retrieval, and antibody incubation parameters:

For tissue preparation:

• Fix tissues in 10% neutral buffered formalin for 24-48 hours

• Process and embed in paraffin following standard protocols

• Section at 4-5 μm thickness onto positively charged slides

For antigen retrieval:

• Heat-induced epitope retrieval is essential for most formalin-fixed tissues

• Citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) heating for 20 minutes at 95-100°C

• Allow sections to cool gradually to room temperature in retrieval solution

For immunostaining:

• Block endogenous peroxidase activity with 3% hydrogen peroxide

• Apply protein block (e.g., 5% normal serum from secondary antibody species)

• Dilute PEDF antibody appropriately (e.g., 1/400 for ab227295 in mouse eye tissue)

• Incubate overnight at 4°C in a humidified chamber

• Wash thoroughly and apply appropriate detection systems (polymer-HRP systems often provide enhanced sensitivity)

• Counterstain with hematoxylin, dehydrate, clear, and mount

Parallel staining of tissues with known PEDF expression patterns serves as an important positive control for protocol validation.

What approaches are recommended for studying PEDF-receptor interactions?

The recent identification of a lipase-linked cell membrane receptor for PEDF (PEDF-R) that binds PEDF with high affinity provides opportunities for studying PEDF-mediated signaling . Several complementary approaches are recommended:

• Co-immunoprecipitation studies:

  • Crosslink PEDF to its receptor using membrane-impermeable crosslinkers

  • Lyse cells under non-denaturing conditions

  • Immunoprecipitate with PEDF antibody (e.g., PEDF Antibody D-10 AC)

  • Analyze precipitates for PEDF-R by Western blotting

• Proximity ligation assays:

  • Fix cells expressing PEDF-R

  • Co-incubate with antibodies against PEDF and PEDF-R

  • Apply oligonucleotide-conjugated secondary antibodies and perform rolling circle amplification

  • Analyze interaction signals by fluorescence microscopy

• Surface plasmon resonance:

  • Immobilize purified PEDF-R on sensor chips

  • Flow purified PEDF at varying concentrations

  • Determine binding kinetics and affinity constants

• Functional validation:

  • Conduct signaling studies measuring downstream effects of PEDF-R activation

  • Employ PEDF-R knockout or knockdown approaches to confirm receptor-dependent effects

These approaches collectively provide a comprehensive understanding of the molecular interactions underlying PEDF's diverse biological activities.

How can researchers develop quantitative ELISA systems for PEDF detection?

Developing a quantitative sandwich ELISA for PEDF requires careful optimization of antibody pairs and assay conditions:

For antibody selection:

• Use a capture antibody with high affinity and specificity (e.g., Polyclonal Anti-Human PEDF)

• Pair with a detection antibody recognizing a different epitope (biotinylated polyclonal antibody at 0.25-1.0 μg/mL concentration)

• Validate antibody pairs for lack of cross-reactivity and interference

For assay development:

• Coat high-binding 96-well plates with capture antibody (typically 1-2 μg/mL) in carbonate buffer (pH 9.6) overnight at 4°C

• Block with 1-2% BSA in PBS for 1-2 hours at room temperature

• Prepare PEDF standards (recombinant protein) in diluent containing carrier protein

• Add samples and standards to the plate

• Incubate with biotinylated detection antibody

• Add streptavidin-HRP conjugate

• Develop with TMB substrate and stop with acid

• Read absorbance at 450 nm

This optimized sandwich ELISA approach allows detection of at least 2000-4000 pg/mL of recombinant human PEDF , making it suitable for quantification in biological samples.

What considerations are important when using PEDF antibodies in neurodegenerative disease research?

When applying PEDF antibodies in neurodegenerative disease research, several specific considerations enhance experimental validity:

• Brain region specificity:

  • PEDF expression varies across brain regions; carefully select relevant regions based on disease pathology

  • Use stereotaxic coordinates for precise sampling in animal models

  • For human samples, document Brodmann areas or anatomical structures precisely

• Cell type resolution:

  • Employ dual immunofluorescence with cell-type markers (neurons, astrocytes, microglia)

  • Consider RNAscope or similar techniques for simultaneous detection of PEDF mRNA and protein

  • Laser capture microdissection may be necessary for cell-type specific analysis

• Disease stage dynamics:

  • Design studies to capture temporal changes in PEDF expression

  • Include presymptomatic, early symptomatic, and advanced disease stages

  • Consider aging controls to distinguish disease effects from normal aging

• Post-mortem considerations:

  • Document and control for post-mortem interval in human studies

  • PEDF stability may vary in different fixation and storage conditions

  • Validate antibody performance in aged, fixed tissues

• Functional correlation:

  • Correlate PEDF immunoreactivity with markers of neuroprotection

  • Consider PEDF fragments that may have distinct activities

  • Integrate findings with known disease mechanisms

These methodological considerations facilitate meaningful interpretation of PEDF changes in the context of neurodegeneration and potential therapeutic applications.

How can researchers resolve common problems with PEDF antibody applications?

When working with PEDF antibodies, several common technical challenges may arise that require systematic troubleshooting:

For specific antibody dilutions, the search results indicate that testing ranges between 1/400-1/1000 for immunohistochemistry and 0.1-0.2 μg/mL for Western blot is appropriate, though optimization for each specific application is essential.

What are the key considerations for selecting between PEDF antibody conjugates?

The choice between various PEDF antibody conjugates should be guided by specific experimental requirements:

• Horseradish peroxidase (HRP) conjugates:

  • Ideal for Western blotting and immunohistochemistry

  • Provides enzymatic amplification of signal

  • Compatible with chromogenic and chemiluminescent detection systems

  • Consider PEDF Antibody (D-10) HRP for direct detection without secondary antibodies

• Fluorescent conjugates:

  • Suitable for immunofluorescence, flow cytometry, and high-resolution microscopy

  • Alexa Fluor® conjugates (PEDF Antibody D-10 AF488, AF546) offer superior photostability

  • FITC and PE conjugates provide options for different filtration systems

  • Consider spectral overlap when designing multiplex experiments

• Agarose conjugates:

  • Optimized for immunoprecipitation studies

  • PEDF Antibody (D-10) AC contains 25% agarose for efficient capture

  • Allows isolation of PEDF and interacting proteins

  • Consider mild elution conditions to preserve protein-protein interactions

• Biotin conjugates:

  • Versatile through interaction with streptavidin systems

  • Provides amplification through multilayered detection

  • Useful in sandwich ELISA development

  • Consider potential interference from endogenous biotin in certain tissues

Each conjugate offers distinct advantages, and selection should be guided by the specific application, instrumentation available, and experimental design requirements.

How are PEDF antibodies contributing to emerging therapeutic applications?

PEDF antibodies are facilitating several promising therapeutic research directions:

• Neovascular disease therapeutics:

  • PEDF's anti-angiogenic properties make it relevant for treating pathological neovascularization in retinal diseases

  • Antibodies help characterize PEDF fragments with enhanced anti-angiogenic activity

  • Monitoring PEDF levels during anti-VEGF therapy provides insights into treatment response mechanisms

• Neurodegenerative disease interventions:

  • PEDF promotes central nervous system neuron survival, protecting against degeneration caused by serum withdrawal or glutamate toxicity

  • Antibodies help track PEDF distribution in the brain after therapeutic administration

  • Immunolabeling enables correlation between PEDF levels and neuroprotection markers

• Cancer therapy development:

  • PEDF's anti-tumorigenic properties suggest potential in oncology

  • Antibodies facilitate screening for small molecules that enhance endogenous PEDF expression

  • Immunohistochemistry with PEDF antibodies helps correlate tumor PEDF levels with prognosis

• Biomarker development:

  • Quantitative ELISA systems using validated antibody pairs enable PEDF measurement in biological fluids

  • Changes in circulating PEDF may serve as biomarkers for disease progression or treatment response

  • Multiplex systems incorporating PEDF detection provide comprehensive biomarker panels

These emerging applications highlight how PEDF antibodies contribute not only to basic research but also to translational medicine and therapeutic development.