PPFIA3 Antibody

What is PPFIA3 and what cellular functions is it associated with?

PPFIA3 (Protein-Tyrosine Phosphatase, Receptor-Type, F Polypeptide-Interacting Protein Alpha-3) is a member of the LAR protein-tyrosine phosphatase-interacting protein (liprin) family. This protein plays crucial roles in synaptic vesicle transport and presynaptic active zone assembly. The protein structure and function are well conserved across both invertebrates and vertebrates, highlighting its evolutionary importance . PPFIA3 contains an N-terminal coiled-coil domain that mediates dimerization processes and interactions with active zone proteins such as RIM and ELKS, while its C-terminal SAM (sterile-α-motif) domain facilitates protein-protein interactions and RNA binding .

What types of PPFIA3 antibodies are available for research applications?

Currently, researchers have access to polyclonal rabbit antibodies against human PPFIA3. Notable examples include rabbit polyclonal antibodies that have been validated for multiple applications including Western Blot (WB), Immunohistochemistry (IHC), Immunoprecipitation (IP), and ELISA . These antibodies demonstrate cross-reactivity with human, mouse, and rat samples, making them versatile tools for comparative studies across species . They are typically supplied in liquid form, purified through antigen affinity methods, and stored in PBS buffer with sodium azide and glycerol at pH 7.3 .

What is the molecular weight of PPFIA3 and how does this impact antibody selection?

PPFIA3 has a calculated molecular weight of approximately 133 kDa based on its 1194 amino acid sequence, though the observed molecular weight in experimental conditions is typically around 140 kDa . This discrepancy between calculated and observed molecular weights can be attributed to post-translational modifications. When selecting antibodies for PPFIA3 detection, researchers should verify that the antibody's detection range includes proteins of this size, particularly when performing Western blot analyses where molecular weight determination is critical for confirming specificity .

What are the optimal dilution ratios for different experimental applications of PPFIA3 antibodies?

Based on validated protocols, the recommended dilution ratios for PPFIA3 antibodies vary by application:

These ratios serve as starting points, and researchers should optimize conditions for their specific experimental systems to obtain optimal results . The sensitivity and specificity of detection can vary significantly between tissue types and experimental conditions, necessitating careful titration during protocol development.

Which buffer systems and antigen retrieval methods are most effective for PPFIA3 immunohistochemistry?

For optimal immunohistochemical detection of PPFIA3, antigen retrieval using TE buffer at pH 9.0 is recommended based on validated protocols. Alternatively, citrate buffer at pH 6.0 may also be used, though potentially with different efficacy . Brain tissues, particularly cerebellum samples from mouse models, have been successfully used for PPFIA3 immunohistochemical analysis. The choice of buffer system significantly impacts epitope accessibility, with alkaline buffers (pH 9.0) often providing superior results for detecting nuclear or membrane-associated proteins like PPFIA3 in fixed tissues.

How should PPFIA3 antibodies be stored to maintain optimal reactivity?

PPFIA3 antibodies should be stored at -20°C where they remain stable for approximately one year after shipment. The storage buffer typically consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . For the 20μl size antibody preparations, manufacturers often include 0.1% BSA as a stabilizer. Unlike some antibody preparations, aliquoting is generally unnecessary for -20°C storage of these particular antibodies, which simplifies laboratory handling procedures while maintaining reactivity .

What are common cross-reactivity issues with PPFIA3 antibodies and how can they be addressed?

While specific cross-reactivity data for PPFIA3 antibodies is limited in the provided sources, polyclonal antibodies generally carry risks of non-specific binding. To address potential cross-reactivity:

• Always include appropriate negative controls in experiments, such as tissue samples known not to express PPFIA3

• Consider pre-absorption with the immunizing peptide when available

• Validate antibody specificity using knockout or knockdown models when possible

• Compare results with alternative antibodies targeting different epitopes of PPFIA3

When working with brain tissues where multiple liprin family members are expressed, blocking protocols should be optimized to minimize background signal from related proteins .

How can researchers validate PPFIA3 antibody specificity in their experimental systems?

Validation of PPFIA3 antibody specificity should follow a multi-faceted approach:

• Western blot verification showing a single band at the expected molecular weight (approximately 140 kDa)

• Comparison of staining patterns with published literature and established expression patterns

• Testing in multiple species if cross-reactivity is claimed (human, mouse, and rat tissues)

• When possible, utilize genetic approaches such as CRISPR knockouts or siRNA knockdown to confirm specific binding

• Consider orthogonal methods such as RNA expression analysis to correlate protein detection with mRNA levels

These validation steps ensure confidence in experimental results and help distinguish between specific signal and background noise in complex tissue samples .

How can PPFIA3 antibodies be utilized in studying neurodevelopmental disorders linked to PPFIA3 mutations?

Recent research has identified rare PPFIA3 variants associated with a syndrome characterized by developmental delay, intellectual disability, hypotonia, autism spectrum disorder, and epilepsy . Researchers investigating these conditions can utilize PPFIA3 antibodies to:

• Compare protein expression levels between patient-derived samples and controls

• Assess subcellular localization changes resulting from pathogenic variants

• Evaluate protein-protein interactions that might be disrupted by mutations in co-immunoprecipitation studies

• Perform immunohistochemical analyses on animal models expressing human PPFIA3 variants to correlate protein distribution with neurological phenotypes

These approaches can provide mechanistic insights into how PPFIA3 variants contribute to neurodevelopmental pathology through altered synaptic function and neuronal connectivity .

What methodological considerations are important when using PPFIA3 antibodies to study synaptic function?

When investigating synaptic function using PPFIA3 antibodies, researchers should consider:

• Co-localization studies with established presynaptic markers such as Bruchpilot (Brp) to assess active zone integrity

• Optimization of fixation protocols to preserve synaptic structures, with paraformaldehyde fixation generally preferred over methanol-based methods

• Combined use of phalloidin staining to visualize muscle structures in neuromuscular junction (NMJ) studies

• Careful selection of secondary antibodies to avoid cross-reactivity when performing multi-label immunofluorescence experiments

• Implementation of super-resolution microscopy techniques to resolve fine details of active zone organization

These methodological considerations are particularly relevant for studies examining the role of PPFIA3 in presynaptic organization and neurotransmitter release mechanisms .

How can researchers effectively use PPFIA3 antibodies in protein interaction studies?

To investigate PPFIA3 protein interactions:

• For immunoprecipitation experiments, use 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate, with optimization required for specific tissue types

• Consider mild detergent conditions (such as 0.5% NP-40 or 1% Triton X-100) to preserve protein-protein interactions

• Include DSP or formaldehyde cross-linking for transient interactions

• Design co-immunoprecipitation experiments to investigate interactions with known binding partners such as RIM, ELKS, and LAR-PTPs

• Validate interactions through reciprocal co-immunoprecipitation using antibodies against suspected binding partners

These approaches can elucidate PPFIA3's role in multiprotein complexes that regulate synaptic architecture and function .

How conserved is PPFIA3 across species and what implications does this have for antibody selection?

PPFIA3's structure and function are highly conserved across both invertebrates and vertebrates, reflecting its fundamental importance in synaptic biology . This conservation has significant implications for antibody selection:

• Antibodies raised against human PPFIA3 often cross-react with mouse and rat homologs, facilitating comparative studies

• Conservation of functional domains (particularly the coiled-coil and SAM domains) means epitopes in these regions may be particularly useful for cross-species detection

• Studies in model organisms such as Drosophila can utilize human PPFIA3 antibodies to detect the fly homolog Liprin-α, though with potential limitations in epitope recognition

• Researchers should validate cross-reactivity experimentally rather than relying solely on sequence homology predictions

Understanding evolutionary conservation can help researchers select antibodies that will work effectively across their chosen experimental models .

What differences in PPFIA3 detection should researchers expect when comparing brain regions or developmental stages?

PPFIA3 expression varies across brain regions and developmental stages, affecting antibody-based detection:

• Cerebellum tissue shows robust detection in immunohistochemical applications, suggesting region-specific expression patterns

• Developmental regulation of PPFIA3 may require adjustment of detection protocols for embryonic, postnatal, and adult tissues

• Expression levels correlate with synaptogenesis, potentially requiring higher antibody concentrations for detection in early developmental stages

• Alternative splicing may generate isoforms with different epitope accessibility across brain regions or developmental timepoints

• Co-expression with other liprin family members varies by region, potentially affecting antibody specificity

Researchers should consider these variables when designing experiments and interpreting results from different neural tissues or developmental stages .

How can PPFIA3 antibodies contribute to understanding synaptic pathology in neurodevelopmental disorders?

PPFIA3 antibodies can be invaluable tools for investigating synaptic pathology in conditions linked to PPFIA3 mutations:

• Immunohistochemical analyses of postmortem brain tissue from affected individuals can reveal alterations in presynaptic architecture

• Patient-derived induced pluripotent stem cells (iPSCs) differentiated into neurons can be immunostained to assess developmental abnormalities in synaptic organization

• Animal models expressing human PPFIA3 variants can be evaluated for changes in protein localization and interaction partners

• Quantitative immunoblotting can determine if pathogenic variants affect protein stability or expression levels

• Super-resolution microscopy with PPFIA3 antibodies can visualize nanoscale changes in active zone morphology

These approaches may reveal how PPFIA3 dysfunction contributes to clinical features such as intellectual disability, autism, and epilepsy observed in affected individuals .

What insights might PPFIA3 antibody studies provide about therapeutic targets for synaptopathies?

Research using PPFIA3 antibodies may identify potential therapeutic targets for synaptopathies:

• Mapping the interactome of PPFIA3 through co-immunoprecipitation followed by mass spectrometry can reveal novel binding partners that might be druggable

• Antibody-based screening assays can identify compounds that stabilize PPFIA3 interactions disrupted by pathogenic variants

• Immunocytochemical analyses following pharmacological interventions can assess restoration of normal PPFIA3 localization

• Identifying compensatory pathways activated in response to PPFIA3 dysfunction could provide alternative therapeutic targets

• Characterizing post-translational modifications of PPFIA3 might reveal regulatory mechanisms amenable to therapeutic modulation

As our understanding of PPFIA3's role in synaptic pathology advances, these approaches may contribute to developing interventions for associated neurodevelopmental disorders .